EMBO/sd-character-level-ner huggingface dataset:
Figure 3(A) Selected frames of time-lapse imaging of control S2 cells or cells treated for 96 hrs with dsRNA against the central portion of Snap29. Note that depleted cell does not display a recognizable metaphase plate, or fail to segregate all chromosomes correctly, or form tripolar spindles or micronuclei (arrowheads; arrowheads in control point to cells dividing normally). (B) Western blotting of extract from control cells and cells depleted of Snap29 (dsSnap29) relative to the experiment in a. (C) Quantification of the mitotic phenotype of control and dsSnap29 depleted cells, based on time-lapse imaging of an average of > 28 individual cells per sample. P value is determined by two-tailed t-test considering all defects together. (D) Selected frames of time-lapse imaging of control U2OS cells and U2OS cells treated for the indicated time. Note that the depleted cell at 48-66h display altered metaphase plate and chromosome segregation (arrowheads). (E) Western blot analysis of extracts relative to the experiments in panel d. (F) Quantification of the time from prophase to entry in anaphase based on analysis of >20 individual cells/sample by time-lapse microscopy. P values are determined by Kruskal-Wallis test with Dunn's multiple comparison analysis. OE: Over-expression.
Metformin suppresses the HSF1-mediated PSR.Figure 1A The connectivity map suggests that metformin generates a gene expression pattern concordant with HSF1 inactivation. Perturbagens inducing and reversing the biological state encoded in the query signature are assigned positive and negative enrichment scores, respectively. B Following treatment with 10 mM metformin overnight, NIH3T3 reporter cells were heat-shocked at 43°C for 30 min and recovered at 37°C for 5 h. GFP induction was quantitated by flow cytometry. HS: heat shock. C Individual proteins in reporter cells treated with and without 10 mM metformin overnight were detected by immunoblotting. D, E Following heat shock and recovery for 4 h, mRNA levels in reporter cells treated with and without 10 mM metformin overnight were quantitated by qRT-PCR (mean ± SD, n = 3, Student's t-test, **P < 0.01, ***P < 0.001). β-actin was used as the internal control. F Reporter cells were treated with 10 mM metformin overnight, and viability was measured using Guava ViaCount® reagents (mean ± SD, n = 5, n.s., not significant, Student's t-test). G Left panel, graphic depiction of the ELISA-based DNA binding assay. HSE: heat shock element. Right panel: Immediately following heat shock at 43°C for 30 min, nuclear proteins of the reporter cells treated with and without 10 mM metformin were extracted for assay (mean ± SD, n = 3, Student's t-test, **P < 0.01, ***P < 0.001). Experimental details are described in Materials and Methods. H Graphic depiction of the adapted PLA. A pair of primary antibodies recognizes DNA-bound HSF1s and dsDNAs, respectively. I HEK293T cells stably expressing a scramble or HSF1-targeting shRNA were treated with 10 mM metformin overnight and heat-shocked at 43°C for 30 min. Cells were fixed for PLA to detect HSF1-DNA interactions. Experimental details are described in Materials and Methods. Scale bars: 50 μm for low magnification and 10 μm for high magnification. J Immediately following heat shock, cytoplasmic and nuclear proteins of the reporter cells treated with and without 10 mM metformin were extracted for immunoblotting. LDH and Lamin A/C were used as cytoplasmic and nuclear markers, respectively. C: cytoplasm; N: nucleus. K Reporter mice (ventral side shown) were pre-treated with metformin for 3 days, 2 mg/mouse/day, via i.p. injection. Following a single i.p. injection of velcade (5 mg/kg) and recovery for 6 h, whole-body luciferase activities were quantitated by bioluminescence imaging. The photon flux of each mouse was expressed as photon/s/cm2/steradian (right panel, mean ± SD, n = 3, one-way ANOVA, *P < 0.05; **P < 0.01).
Lurbinectedin inhibits SCLC growth.Figure 5Dose response of A549 (black), NCI-H460 (yellow), NCI-H510A (blue), NCI-H82 (red) and DMS-53 (green) cell lines after treatment with (A) Carboplatin, (B) Etoposide, (C) Topotecan (D) Lurbinectedin Data representing the half maximal inhibitory concentration (IC50) expressed in molar concentrations. Cell survival is shown in percentage and 50% threshold is highlighted in dotted line. The data (presented as mean ± SEM) are the average of three independent experiments. Dose response of A549 (black), NCI-H460 (yellow), NCI-H510A (blue), NCI-H82 (red) and DMS-53 (green) cell lines after treatment with Cisplatin. Data representing the half maximal inhibitory concentration (IC50) expressed in molar concentrations. Cell survival is shown in percentage and 50% threshold is highlighted in dotted line. The data (presented as mean ± SEM) are the average of three independent experiments. (F) Venn diagrams showing the overlap between genes downregulated after 50nM treatment with lurbinectedin (Blue circle) and 20uM treatment with cisplatin (yellow circle) in RNAseq. (G) RT-PCR showing the downregulation of ASCL1, NEUROD1, BCL2, INSM1, MYC, MYB, AURKA and CDK7 genes after 50nM lurbinectedin treatment (light blue bars), 50nM or 20uM Cisplatin (CsPt; Light yellow and dark yellow bars, respectively) as compared to untreated samples (Ctl- Dark blue bars). Data is represented as Relative mRNA Expression normalized to Ctl. Data is presented as Mean ± SD. (n=2 biological replicates).
Figure 4(A) Schematic representation of DNGR-1, Dectin-1 and the chimeric protein consisting of the neck region of DNGR-1 and CTLD of Dectin-1. (B) Supernatants containing ECD of long mouse DNGR-1, Dectin-1 and the chimeric receptor were prepared for SDS-PAGE and Western blot under non-reducing conditions. (C) Supernatants containing indicated proteins were diluted into PBS or pH 6.1 MES buffer and analyzed using SDS-PAGE and Western blot under reducing conditions. HRP-conjugated anti-FLAG antibody was used for detection of all proteins. Numbers on the side of blots indicate positions of molecular weight markers. Reduction-resistant dimers are indicated with an * and reduction sensitive protein with a +.
CTNS-deficient cells are more susceptible to oxidative stress-induced cell death.Figure 9WT and Ctns−/− mouse fibroblasts were treated with either 50 μM H2O2 or 1 mM paraquat (PQ) for 4 h and stained with (A) FITC-annexin V or (B) propidium iodide (PI) and analyzed by FACS. Results from 3 different experiments are shown as mean ± SEM. In (A), *P = 0.05; ***P = 0.0002. In (B), *P = 0.046; **P = 0.0065. Unpaired t-test. WT and Ctns−/− cells were treated as described above and samples analyzed by Western blot using antibodies recognizing cleaved (active) caspase 3. Data are representative of 2 independent experiments with similar results.
Figure 3A) Immunoblot analyses showing MAPK phosphorylation after flg22 induction in Col-0 and in mkkk7. Protein extracts were made from seedlings treated with 1 μM flg22 and samples were taken at t=0, 10 and 30 min post induction. The p44/42 antibody was used to detect phosphorylated MAPKs. Position of the individual phosphorylated MAPKs is indicated at the right. Equal loading of proteins is shown with an α-Actin antibody as a loading control (bottom panel). Three biological replicates were done with identical results B) MPK6 phosphorylation is specifically enhanced in mkkk7. Comparison of phosphopeptide abundances from selected MAP kinases in Col-0 (blue) and mkkk7 (red) seedlings at t = 0 min and t = 10 min after 1 μM flagellin treatment by selected reaction monitoring (SRM) mass spectrometry. Phosphopeptide corresponding to MKKK7 are only detectable in Col-0 seedlings and are non-detectable (ND) in mkkk7. Bars represent means of measured peptide areas (sum of all transition areas) for three biological replicates, with error bars ± SEM (n=3). Asterisks indicate significant difference between Col-0 and mkkk7 at individual time points (student t-test, *>0.05, **>0.01 and ***>0.001). ND indicates integration of an area without transitions significantly above background. Above each graph the protein name and the phosphorylated residue (in brackets) is indicated as well as the corresponding phosphopeptide sequence. Serine (S), Threonine (T) or Tyrosine (Y) phosphorylation is indicated by "[+80]".
Figure 7A-F Live confocal images of ddaC neurons at WP stage and 16 h APF. stai RNAi neurons (B), staiSK7 (C), staiKO (D) mutant MARCM clones showed similar dendrite pruning defects, compared to the wild-type ddaC neurons (A). Double RNAi of stai and efa6 significantly enhanced the dendrite pruning defects (E), compared to their individual RNAi knockdown. Expression of stai RNAi in efa612/GX6w- mutant background also significantly enhanced the dendrite pruning defects (F), compared to the RNAi or mutant alone. Red arrowheads point to the ddaC somas. G-H Quantification of dendrite severing defects and unpruned dendrite lengths at 16 h APF. I-L Representative kymographs of EB1-GFP comets driven by Gal44-77 in the proximal dendrites of ddaC neurons at 96 h AEL. M-P Quantification of EB1-GFP comet orientation, number, track length and speed.
Figure 4Volcano plot of ERα dependent miRNA expression in BM67 cells quantified by small RNAseq. Down- and up-regulated miRNAs (FDR<0.2 and fold-change > 1.25) are colored in blue and red, respectively. Validation of miRNA expression using qPCR. Normalized expression (mean ± standard deviation; n = 3) (B) and expression fold change (shERα vs. shCtrl) obtained from small RNAseq vs. qPCR (C). Percentage of miRNA among small RNAs in shERα and shCtrl BM67 cells (mean ± standard deviation; n = 3). Assessment of the relationship between presence of miRNA target sites and translational offsetting. (E) For visualization, mRNAs were assigned angles describing their mode of regulation such that mRNAs with congruently modulated total and polysome-associated mRNA obtain an angle of ~45 degrees, whereas offset mRNAs are assigned an angle of ~0 degrees. (F) The distributions (quantified by kernel density estimation) of such angles were then compared between mRNAs whose levels were induced upon ERα depletion, which were also targets of each suppressed miRNAs separately (blue lines); and the set of background transcripts (mRNAs induced upon ERα depletion but not targets of suppressed miRNAs; black). (G) For such subsets of miRNA targets (i.e. from F), the relative number of offset and non-offset mRNAs (as determined by anota2seq analysis) was also compared to the background (same as in F) using Fisher's exact test; and the resulting adjusted p-values (FDRs; across all assessed miRNAs) were visualized as a histogram. (H-I) A similar assessment as in (F-G) but for suppressed mRNAs and induced miRNAs. (J) A similar assessment as in (H) but for randomly selected groups of 10 miRNAs (gray lines) compared to background (black) and targets of all induced miRNA (red; i.e. from H).
Figure 7A. A diagram of experimental design. 8 to 12-week-old PbkKI/KI and PbkWT/WT mice were treated with MI-463 at 70mg/Kg (n=4) or vehicle (n = 4) by gavage daily. B, GTT data from PbkKI/KI and PbkWT/WT mice (n=4 for each group) after 8 weeks of MI-463 or vehicle treatment (B) (***P < 0.0001 (Two-way ANOVA), ns, not statistically significant difference) and AUC of the GTT results Dot line, fasting blood glucose number >150 was designated as hyperglycemia. (C) (***P = 0.0005 (two-tailed unpaired student's t-test). ns, not statistically significant difference). The mice were starved overnight prior to the test (16h). D. In vivo GSIS test on 11-week-old male PbkKI/KI and PbkWT/WT mice (n=4 for each group). The mice were starved overnight prior to the test (16h). Y-axis represents the ratio of insulin level at 15 min over insulin level at 0 min with glucose challenge. **P = 0.0042 (two-tailed unpaired student's t-test). ns, not statistically significant difference. E-L. Representative images of insulin (green) and Ki67 (red) double staining on mouse islets with 8 wks MI treatment (E-H), and insulin (green) or Pbk (red) double staining on mouse islets (I-L). Nuclei were labeled by DAPI (blue). Islet area was circled by white dashed line. Ki67 and Pbk staining are denoted by white arrows. Scale bars: 50 μm. M, N. Quantification of the percentages of Ki67 staining-positive beta cells (M) and Pbk staining-positive beta cells (N). Four mice for each group, and 5-10 islet images per mouse were analyzed. **P = 0.0026 (M, WT/WT-Mock : WT/WT-MI-463), **P = 0.0013 (M, KI/KI-Mock : KI/KI-MI-463), **P = 0.0011 (N, WT/WT-Mock : WT/WT-MI-463), ***P = 0.0009 (N, KI/KI-Mock : KI/KI-MI-463) (two-tailed unpaired student's t-test). ns, not statistically significant difference. O. Beta cell mass comparison between MI-463- and vehicle-treated PbkKI/KI mice and PbkWT/WT mice (n=4 for each group). *P = 0.0174 (two-tailed unpaired student's t-test). ns, not statistically significant difference.
KRIT1 loss-of-function activates the mTOR-ULK1 pathway.Figure 2Immunoblot analysis with antibodies directed against phosphorylated mTOR (Ser 2448), total mTOR, phosphorylated p70 S6 Kinase (Ser 371), total p70 S6 Kinase, phosphorylated 4E-BP1 (Thr 37/46), and total 4E-BP1; actin was used as a loading marker. Where indicated, KRIT1 wt and KRIT1-KO endothelial cells were treated with 100 nM Torin1 for 4 h. The results are representative of three independent experiments. Immunoblot analysis of total ULK1 and actin in KRIT1 wt and KRIT1-KO endothelial cells. Where indicated, cells were treated with 100 nM Torin1 for 4 h. The results are representative of three independent experiments. Immunoblot analysis of p62, LC3 I/II, and actin in KRIT1 wt and KRIT1-KO endothelial cells treated with 100 nM Torin1 or 500 nM rapamycin for 4 h. The results are representative of three independent experiments. Immunoblot analysis with antibodies directed against phosphorylated mTOR (Ser 2448), total mTOR, phosphorylated p70 S6 Kinase (Ser 371), total p70 S6 Kinase, phosphorylated 4E-BP1 (Thr 37/46), and total 4E-BP1; actin was used as a loading marker. Where indicated, KRIT1-KO re-expressing KRIT1 (KO+KRIT1) and KRIT1-KO MEFs were treated with 100 nM Torin1 for 4 h. The results are representative of three independent experiments. Immunoblot analysis of phosphorylated ULK1 (Ser 757), total ULK1, and actin in KRIT1 KO+KRIT1, and KRIT1 KO MEFs. Where indicated, cells were treated with 100 nM Torin1 for 4 h. The results are representative of three independent experiments. Immunoblot analysis of p62, actin, LC3 I/II in KO+KRIT1 and KRIT1-KO cells. Where indicated, cells were treated with 100 nM Torin1 for 4 h or 500 nM rapamycin for 4 h. The results are representative of three independent experiments. KRIT1 wt and KRIT1-KO endothelial cells were transiently transfected with mRFP-GFP-LC3. Where indicated, the cells were treated with 100 nM Torin1 for 4 h or 2 μM xestospongin B for 4 h. The differences in the autophagic flux were evaluated by counting the yellow LC3 I/II dots/cell (RFP+GFP+) and red LC3 dots/cell (RFP+GFP−) for each condition. Yellow dots: autophagosomes; red dots: autophagolysosomes. *P = 5.74e−5 (red dots, WT ctrl vs. WT Tor1); *P = 9.62e−5 (red dots, WT ctrl vs. WT xesto); *P = 0.00727 (red dots, WT ctrl vs. KO ctrl); #P = 0.00046 (red dots, KO ctrl vs. KO Tor1). The data are expressed as the mean ± s.e.m. KO+KRIT1 and KRIT1-KO MEFs were transiently transfected with the mRFP-GFP-LC3 tandem construct. Where indicated, the cells were treated with 100 nM Torin1 for 4 h or 2 μM xestospongin B for 4 h. The differences in the autophagic flux were evaluated by counting the yellow LC3 I/II dots/cell (RFP+GFP+) and red LC3 dots/cell (RFP+GFP−) for each condition. Yellow dots: autophagosomes; red dots: autophagolysosomes. *P = 0.00023 (red dots, KO+KRIT1 ctrl vs. KO+KRIT1 Tor1); *P = 0.00045 (red dots, KO+KRIT1 ctrl vs. KO+KRIT1 xesto); #P = 3.08e−6 (red dots, KO ctrl vs. KO Tor1); ##P = 6.73e−5 (yellow dots, KO+KRIT1 ctrl vs. KO ctrl). The data are expressed as the mean ± s.e.m. of four independent experiments.
Figure 5qRT-PCR for mDia2/DIAPH3 relative to RPL27 using RNA from normal human skin (N=11), SCCs (N=9) and BCCs (N=7). Representative skin/tumor sections from normal human skin, SCC and BCC patients stained for mDia2/DIAPH3. Note the strong mDia2/DIAPH3-positivity in the tumor stroma. Scale bars: 100 μm. D: Dermis, E: Epidermis: T: Tumor. Expression levels of mDia2/DIAPH3 in tumor and normal tissues based on TCGA cancer data (TCGA data version 20141017, http://firebrowse.org/). Gene expression levels were presented as normalized log2 RSEM (RNA-Seq by Expectation-Maximization) index from the database. BLCA: Bladder Urothelial Carcinoma (N = 408 & 19 tumor and normal tissue samples, respectively), BRCA: Breast Invasive Carcinoma (N = 1100 & 112), CESC: Cervical SCC (N = 306 & 3), ESCA: Esophageal SCC (N = 185 & 11), HNSC: Head-Neck SCC (N = 522 & 44), LIHC: Liver Hepatocellular Carcinoma (N = 373 & 47), LUSC: Lung SCC (N = 501 & 51), PAAD: Pancreatic Adenocarcinoma (N = 179 & 4), PRAD: Prostate Adenocarcinoma (N = 496 & 50), READ: Rectum Adenocarcinoma (N = 167 & 10), STES: Stomach and Esophageal Carcinoma (N = 600 & 46), UCEC: Uterine Corpus Endometrial Carcinoma (N = 546 & 35). Box plot shows relative mDia2/DIAPH3 expression in human tumor (red) and normal (blue) tissue. Boxes indicate the interquartile range, whiskers indicate data points within 1.5 times interquartile range, dots outside the whiskers indicate outliers, and the central line indicates median. Relative gene expression of mDia2/DIAPH3 and INHBA in the stroma of intrahepatic cholangiocarcinoma, breast and colorectal cancers vs. stroma of respective normal tissues based on datasets GSE45001 (D) (N = 10 normal tissue & 10 cancer samples, GSE9014 (E) (N = 4 & 13), and GSE35602 (F) (N = 6 & 53), and in CAFs from breast cancer samples vs. normal breast fibroblasts (GSE29270 dataset) (G) (N = 5 & 23). NS: Normal stroma, CS: Cancer stroma, NF: Normal fibroblasts, CAF: Cancer-associated fibroblasts). The expression values of the datasets were obtained using GEO2R tool in the GEO database. Kaplan-Meier survival curves based on datasets from GEO, EGA and TCGA, demonstrating that high expression of both mDia2/DIAPH3 and INHBA in patients with liver, stomach and breast cancer correlates with poor overall survival. Data and statistical analyses were collected from KMPlotter.
Figure 3(A)-(C) Previously reported modifiers. Upper panels: Scatter plots associating the expression of the GCD (SMN1) vs. identified PMs in healthy GTEx muscle tissues (left panels) and spinal cord tissues (right panels); the expression of SMN1 (X-axis) vs. that of (A) HNRNPU, (B) SF1 and (C) SRSF4. Bottom Panels: Boxplots associating the expression of the identified DPMs in case-control studies; the expression of (A) HNRNPU, (B) SF1, and (C) SRSF4 in SMA and in healthy control muscle (left panels) and spinal cord (right panels) tissues. Data information: Empirical P-values significance is indicated for two thresholds (** is P-value<0.01 and *** is P-value<0.001, using the permutation test are for the hyper-geometric enrichment test. There are six control (blue) and eleven SMA biological replicates (red) from muscle tissues and four control (blue) and six SMA biological replicates (red) from spinal cord tissues. The Reads Per Kilobase Million (RPKM) measure was used in the GTEx dataset, and the Fragments Per Kilobase Million (FPKM) measure was used in the SMA case-control dataset. (D)-(E) Candidate modifiers. Upper panels: Scatter plots associating the expression of the GCD (SMN1) vs. identified PMs in healthy GTEx muscle tissues (left panels) and spinal cord tissues (right panels); the expression of SMN1 (X-axis) vs. that of the negative DPM modifiers (D) U2AF1 and (E) HNRNPA0. Bottom Panels: Boxplots associating the expression of the identified DPMs in case-control studies; the expression of (D) U2AF1 and (E) HNRNPA0 in SMA and in healthy control muscle (left panels) and spinal cord (middle panels) tissues. Right panels show the levels of these predicted modifiers in healthy muscle samples with low ratio of SMN2-FL to SMN2∆7 and those with high ratio of SMN2-FL to SMN2∆7. Data information: Empirical P-values significance is indicated for two thresholds (** is P-value<0.01 and *** is P-value<0.001, using the permutation test The P-values denoted in the upper panels of and E are for the hyper-geometric enrichment test. There are six control (blue) and eleven SMA biological replicates (red) from muscle tissues and four control (blue) and six SMA biological replicates (red) from spinal cord tissues. The Reads Per Kilobase Million (RPKM) measure was used in the GTEx dataset, and the Fragments Per Kilobase Million (FPKM) measure was used in the SMA case-control dataset.
Figure 4Immunofluorescent images of liver and kidney 24h after sham (with or without starvation) or CLP (n= 6-7/group, combined data of 2 independent experiments). White scale bar = 20µm. Cryosections were stained with Acti-stain (green), Hoechst (blue), and LipidTOX (red). Z-stacks were generated in 5-10 areas scattered across the entire tissue section. Immunofluorescent images of liver and kidney 24h after sham (with or without starvation) or CLP (n= 6-7/group, combined data of 2 independent experiments). White scale bar = 20µm. The amount of lipid droplets (LDs)/cell and average size of LDs (represented by voxel counts) were calculated for each Z-stack. Averages of the amount and size of LDs were converged for each mouse and biological replicates are depicted in the table as mean ± SEM. Quantification of lipid peroxidation by determination of (C) MDA and (D) 4-HNE concentrations in liver homogenates 24h post-surgery in sham and CLP mice as described in methods (n = 6-7/group, combined data of 2 independent experiments). P-values were calculated with 1-way ANOVA tests. Apoptosis in liver paraffin-fixated sections 24h after sepsis, measured by TUNEL staining, and presented as % PI positive cells/µm² tissue area. (n= 6/7 mice/group, combined data of 2 independent experiments). P-values were calculated with 1-way ANOVA tests.
Figure 1Growth suppression in cancer cells after 72h of treatment with APR-246 +/- MRP1 inhibitor MK-571 as shown by the WST-1 assay (n ≥ 3, n shown in Table S1). Cross in heatmap indicates no data available. Growth suppression in human dermal fibroblasts (HDF) by WST-1 assay after 72h of with APR-246 +/- MK-571 (n = 3). Mean IC50 values (µM) for APR-246 +/- 20 μM MK-571 of 21 cell lines. *p < 0.0001, Wilcoxon test. Each dot represents one cell line. Mean IC50 values and n shown in Table S1. ZIP synergy scores of most synergistic area sub-grouped according to TP53 gene status for 21 cell lines. Each dot represents one cell line. Scores and n are shown in Table S1. Sub-G1 DNA content of OVCAR-3 cells (R248Q) after APR-246 +/- MK-571 treatment as assessed by propidium iodide staining at 48h (n = 3). OVCAR-3 cell confluency by IncuCyte® during 72h of treatment with APR-246 +/- MK-571 (n = 3). Growth suppression in HCT116 WT (n = 4) and R248W cells (n = 3) after 72h treatment with APR-246 +/- MRP1 inhibitor Reversan, as shown by WST-1. Western blot analysis of MRP1 (Cell Signaling.), p53 (DO-1) and GAPDH of HCT116 WT and R248W cells 48h after transfection with negative control siRNAs or siRNAs targeting MRP1 or p53. Growth suppression (WST-1 assay) of HCT116 WT and R248W cells transfected with MRP1 siRNA (n ≥ 5 and n ≥ 2 respectively) after 48h APR-246 treatment. Indicated values are average of four individual siRNAs targeting MRP1 and two individual negative control siRNAs. Data and n of individual siRNAs are shown in Fig S1J, data is also part of Fig 7B. Western blot analysis of MRP1 (Cell Signaling.), p53 (DO-1) and GAPDH of HCT116 WT and R248W cells 72h after transfection with empty vector (EV) and MRP1 expression vector. Growth suppression (WST-1 assay) of HCT116 WT and R248W cells transfected with empty vector (EV) and MRP1 vector after 72h APR-246 treatment (n = 4). Data is also part of Fig S7E.