EMBO/sd-character-level-ner huggingface dataset:
sgRNA integration and transcription activation of ABCB1A) TLA analysis for ABCB1 contacts in RPE-1 parental and Taxol resistant clone sg6C9 covering the whole genome.Figure 2A) TLA analysis for ABCB1 contacts in RPE-1 parental and Taxol resistant clone sg6C9 covering the whole genome. Green arrow in sg6C9 shows a de novo interaction found between ABCB1 and a region in chromosome 6. B) TLA analysis for RPE-1 Parental and sg6C9. Zoom in in the region of chr6 with de novo interaction for sg6C9. Image modified from IGV viewer. C) Graphical representation of the PCR products to assess vector integration. Two different primer pairs were used to PCR the vector integration: 1) a common EEF1A1 Forward (F) primer with a specific Reverse for each break site (5/11/6/3/12R). Only when EEF1A1 is integrated in cis we will obtain a PCR product. 2) A forward and Reverse primer to amplify each specific break site (5/11/6/3/12F and R). If EEF1A1 is integrated in the break site, the PCR product will be bigger. D) PCR products using the primers in C(1) over the ABCB1 and EEF1A1 regions in RPE-1 Parental and the different Taxol resistant clones. E) PCR products using the primers in C(2) over the specific break site in the ABCB1 promoter in RPE-1 Parental and the different Taxol resistant clones. F) TLA analysis for RPE-1 Parental and sg6C9. Reads are aligned to the lenti-guide vector. The location of the EF1α promoter from the vector is highlighted in red. Image modified from IGV viewer. G) Percentage of RPE-1 cells targeted with the ABCB1 promoter sgRNAs found with vector integration. Percentages were calculated from data in Fig 1A. Each dot represents a CFA replicate experiment. Horizontal bars represent the mean of lentiviral integratation per sgRNA.
IKKε expression in breast cancer is associated with immune infiltration.Figure 3IKKε expression was assessed semi-quantitatively using a 0-3 grade scale ("0": no staining; "1": weak; "2": moderate; "3": strong) on human breast cancer sections using immunohistochemistry. Representative images according to the IKKε staining grade are shown. Scale bar: 100 μm. Distribution table of IKKε expression in the cohort of 66 human breast carcinomas according to the staining grade. Correlation between IKKε expression and immune cell infiltration ("0": no inflammatory cells; "1": weak; "2": moderate; "3": strong; "4": very strong). Bubble plot of IKKε and immune infiltration grades, size shows the number of tumours falling in the category. Spearman's rho coefficient and significance of difference from slope = 0 (p) are shown. Correlation between IKBKE mRNA levels (log2 ratio) and immune signature (Yoshihara et al, 2013) in the METABRIC transcriptomic dataset from 1981 breast cancer patients (Curtis et al, 2012). Pearson's correlation rho coefficient and significance of difference from slope = 0 (p) are shown. IKBKE gene copy number and mRNA levels (log2 ratio) from the METABRIC transcriptomic dataset (see D) are plotted. Estimated +1 copy number values are shown by the dotted vertical line. No significant correlation by Pearson's correlation rho coefficient and significance of difference from slope = 0 (p) was detected.
Figure 3Anti-Ser259-MYRF antibody was generated using the peptides containing pS259. Western blotting analysis of the corpus callosum in PLP-23Q or PLP-150Q mice with anti-pS259 and an antibody to total MYRF. Note that LAQ (5 mg/kg) treatment for 2 months decreased the phosphorylation in both full length (fMYRF) and N-terminal MYRF (nMYRF) in PLP-150Q mice. Ratios of phosphor-S259-MYRF (pMYRF) and total MYRF to GAPDH. The ratios were obtained from 3 independent western blotting experiments. Student's t-test; ***P=0.0007. Data are mean ±SEM. Anti-Ser259-MYRF immunohistochemical staining of the corpus callosum (CC, indicated between two dotted lines) of PLP-23Q and PLP-150Q mice showing decreased expression of Ser259-MYRF by LAQ (5 mg/kg) treatment for 2 months. Micrographs at 10X (upper) and 40X (lower) are shown. Scale bars: 40 μm. Quantitative statistics of the cells stained by anti-Ser259 MYRF in each filed was presented. Twenty random fields (40X) of each section were examined to count anti-Ser259 positive cells, n=3 mice in each group. One-way ANNOVA with Tukey's test. ***P=0.0003; **P=0.0039. Data are mean ±SEM. Information of postmortem brains of control and HD patients. Western blotting analysis of MBP and Ser259-MYRF expression in the postmortem brain cortex tissues in 4 HD patients and 4 non-HD control individuals. Ratios of MBP to GAPDH, or Ser259 (pMYRF) to total MYRF are presented on the right. One-way ANNOVA with Tukey's test. MBP: ***P=0.0004; pMYRF: **P=0.0051. Data are mean ±SEM. Western blotting analysis of the expression of Ser259 (pMYRF) and total MYRF (full-length: fMYRF and N-terminal: nMYRF) in 13-months-old HD-KI and WT mouse cortex. Ratios of pMYRF to total MYRF obtained from 3 independent experiments were presented beneath the blots. One-way ANNOVA with Tukey's test. ***P=0.00083. Data are mean ±SEM.
Figure 5(A) Fibroblasts from three individual patients with confirmed C9orf72 repeat expansions were investigated. (B) Knockdown of hnRNPA3 with two independent siRNA results in selective depletion of hnRNPA3 in fibroblasts from 3 C9orf72 repeat carriers. Actin was used as a loading control. (C) Knockdown of hnRNPA3 increases RNA foci in cells derived from C9orf72 carriers. No G4C2 repeat RNA foci are detected in fibroblast without C9orf72 repeat expansions (WT). Nucleoli were stained with anti nucleolin antibodies (green) and nuclei were stained with DAPI (blue). (D) Quantification of the relative frequency of RNA foci positive cell (RNA foci positivity) (fold change). n=3 experiments for each case. (E) Quantification of the number of RNA foci per RNA foci-positive cell (fold change). n=3 experiments for each case. (D, E) The average foci number of non-treated (N/T) fibroblast was normalized to 1 in each case. Bars indicate mean. Individual points indicate mean values obtained from a single experiment (49-214 cells were counted in single experiment). Error bars indicate 95%CI, ANOVA, Tukey-Kramer HSD, *p<0.05, **p<0.01, ***p<0.001. (F) Representative presentation of increased RNA foci in C9orf72 carriers upon lentiviral-mediated hnRNPA3 knockdown. (G) Quantification of the number of RNA foci (fold change). 3 cases were analyzed. Single points indicate an average obtained from 29-30 cells per case. Color code labels values obtained in fibroblasts derived from the three individual patients. Two-tailed t-test. Mean ± SEM. Scale bar= 10 μm.
Figure 2Atomic structures of T4 proteins are shown as ribbon diagrams of various colors and labeled with T4 and A511 gene product names as well as with their functional orthologs common to all contractile-like systems (e.g. BH1, see Table 2). The putative endopeptidase domain of A511 gp98 is modeled by the crystal structure of RipA of Mycobacterium tuberculosis (colored purple). Red lines and labels in panel (A) indicate the position of end-on views shown in panels (B through F) (looking from the capsid).
expression.Figure 8(A) Purified mitochondrial preparation from multiple HKC strains from White and Black donors (12 per ancestry; passage 2,3; triplicate cultures) were analyzed for levels of Electron Transport Chain activity (ETC, nmol of reduced cytochrome c/min/mg protein), ATP (nmol/mg protein) and ROS (MtROS (nmol/mg mitochondrial protein). In parallel, Acid Soluble Metabolites (ASM, pmol/h/mg protein) levels were measured as a readout of β-oxidation. Similar measurements of HKCs strains at a later passage are shown in Appendix Fig S6. Data are displayed as average values for all tested HKC strains (3 cultures per strain for ETC, ATP and FAO, 1 culture per strain for mtROS, white and black dots, depending on ancestry) together with mean +/- SD. n(HKC strains per ancestry)= 12 ***p<0.0005; ****p<0.0001, 2-tailed unpaired t-test. Individual experimental values are provided in Dataset EV3. (B) Multiple HKC strains (249W, 260W, 275W) stably transduced with an HSD17B7-expressing lentivirus (HSD-oe) versus empty vector control (Ctrl) were analyzed as in (A). Data are shown as individual values for 3 parallel cultures per strain together with mean +/- SD. *p<0.05; **p<0.01; ***p<0.0005; 2-tailed unpaired t-test. Individual experimental values are provided in Dataset EV3. (C, D) SCC13 cells stably infected with an HSD17B7-expressing lentivirus (HSD-oe) versus empty vector control (Ctrl) (C) or two HSD17B7-silencing lentivirus (shHSD2, shHSD4) versus vector control (shCtrl) (D) were analyzed as in the previous panels. Shown are individual values for 3 parallel cultures per condition together with mean +/- SD. **p<0.005; ***p<0.0005; ****p<0.0001. 2-tailed unpaired t-test. Additional metabolic measurement experiments of SCC cells plus/minus HSD17B7 overexpression and silencing, in the context of additional manipulations are shown in Fig 9.
Fig.Figure 4A, B) ELISA of the (A) RIPA soluble fraction of female APP/PS1 and APP/PS1 PD-1-/- mice for Aβx-40 (biological replicates with n=6 for APP/PS1 and n=5 for APP/PS1 PD-1-/-, mean+/-SEM, one-way ANOVA (DF = 1, F = 0.6, p=0.45), Tukey's HSD, ** p<0.01, *** p<0.001) and (B) of the SDS soluble fraction (biological replicates with n=6 for APP/PS1 and n=5 for APP/PS1 PD-1-/-, mean+/-SEM, one-way ANOVA(DF=1, F= 32.8, p=2x10-5), Tukey's HSD, ** p<0.01, *** p<0.001). C) Thioflavin T histochemistry from sagittal brain sections in the hippocampus (Hc), and cortex (Cx) of female APP/PS1 and APP/PS1 PD-1-/- mice. Nuclei were stained using Hoechst 33342 (bar = 500 μM). D, E) Mice were analyzed for (D) percentage of the covered areas (5 sections per mouse analyzed with the mean representing an individual data point; biological replicates with n=6 for APP/PS1 and n=9 for APP/PS1 PD-1-/-, mean +/-SEM, one-way ANOVA (DF=1, F=0.905, p=0.90), Tukey's HSD, ** p<0.01, *** p<0.001) and (E) the number of plaques per mm2 (5 sections per mouse analyzed with the mean representing an individual data point; biological replicates with n=6 for APP/PS1 and n=9 for APP/PS1 PD-1-/-, mean +/-SEM, one-way ANOVA (DF=1, F=4.08, p=0.054), Tukey's HSD, ** p<0.01, *** p<0.001). F) Western blot analysis of brain lysates from female APP/PS1 and APP/PS1 PD-1-/- mice for the expression of APP and APP C-terminal fragments using antibodies 6E10 (transgene expression) and CT15 (transgene and endogenous expression). Tubulin was used as a loading control. G) Time needed to reach the hidden platform (latency in seconds) in the Morris water maze test (mean +/-SEM of biological replicates with n=12 for wt, n=5 for PD-1-/-, n=7 for APP/PS1, and n=8 for APP/PS1 PD-1-/-, one-way ANOVA (DF=3, F=17.72, p=1x10-9), Tukey's HSD, * p<0.05, *** p<0.001). H) Analysis of the area under the curve (mean +SEM of biological replicates with n=12 for wt, n=5 for PD-1-/-, n=12 for APP/PS1, and n=8 for APP/PS1 PD-1-/-, ANOVA (DF=3, F=6.98, p=0.001), * p<0.05, ** p<0.01).
Figure 6A Hypothetical signaling pathway for the induction of hyperactive Cdk5 induced by repression of VPS34 downstream of neuronal activity. B Cdk5 inhibition by Roscovitine (Ros, 10 µM) rescues the progressive accumulation of Synaptophysin-pHluorin on the neuronal surface induced by VPS34IN1 (10 µM). N = 4 independent experiments (20 images per condition); Two-way ANOVA; Tukey's Multiple Comparisons Test. Data information: Data presented as mean ± SEM n.s. not significant; *p < 0.05; **p < 0.01; ***p < 0.001 C Calcineurin inhibition by Cyclosporin A (10 µM) phenocopies VPS34IN1-induced loss of PI(3) P with respect to the accumulation of Synaptophysin-pHluorin on the neuronal surface after four successive stimulation trains (40 Hz, 5 s). Data were analyzed as in B. N = 3 independent experiments (15 images per condition); Two-way ANOVA; Tukey's Multiple Comparison Test. Data information: Data presented as mean ± SEM n.s. not significant; *p < 0.05; **p < 0.01; ***p < 0.001 D Basal excitatory synaptic transmission of CA3-CA1 connections in acute hippocampal slices treated with DMSO (0.03%), Roscovitine (Ros, 10 µM) or VPS34IN1 (3 µM) plus Roscovitine. Representative fEPSP traces (above) and the relative slope of fEPSPs (below) are shown. Concomitant application of VPS34IN1 and Roscovitine facilitates excitatory synaptic transmission, akin to Roscovitine alone, and in contrast to VPS34IN1 alone N = 6 slices per condition from 6 mice; Student's t-test. Data information: Data presented as mean ± SEM n.s. not significant; *p < 0.05; **p < 0.01; ***p < 0.001 E Decreased paired paired-pulse facilitation (PPF) at hippocampal CA3-CA1 synapses treated with Roscovitine alone (Ros, 10 µM) or VPS34IN1 (3 µM) plus Roscovitine. Representative traces of fEPSPs with a 20 ms interpulse interval is shown above. The ratio of the slope of the second to the first response over a range of interstimulus intervals (10-500 ms) is plotted below. The data show decreased facilitation of the second response in Roscovitine or VPS34IN1 plus Roscovitine-treated acute hippocampal slices, unlike VPS34IN1 alone . N = 6 slices per condition from 6 mice; Two-way RM ANOVA. Data information: Data presented as mean ± SEM n.s. not significant; *p < 0.05; **p < 0.01; ***p < 0.001
a) We performed filter-trap assays to determine the extent of accumulation of insoluble AR protein in NPC lines from three different controls and three different SBMA patients.figf7(a) We performed filter-trap assays to determine the extent of accumulation of insoluble AR protein in NPC lines from three different controls and three different SBMA patients. Significantly higher levels of insoluble AR protein were present in the SBMA NPC lines, as based on densitometry; n = 3 independent experiments, P 0.05, t-test, t(4) = 4.026. AU, arbitrary units. (b) NPCs from three different controls and three different SBMA patients were cultured in the presence of JC-1 dye. Results are presented as a Tukey box plots with box corresponding to first to third quartiles from the median and whiskers corresponding to 1.5 quartiles from the median; n = 3 independent experiments, *P 0.05, t-test, t(4) = 3.983. (c) We transfected NPC lines derived from SBMA patients and controls (three different NPC lines per patient) with the mCherry-EGFP-LC3 vector. Autophagic vesicles were detected as colored puncta, as shown. Scale bar, 10 μm. (d) Quantification of autophagic vesicle type from c. SBMA NPCs exhibit a significant increase in autophagosomes; n = 18 different samples: 3 clones per line, 3 lines per group, *P 0.05, t-test; t(18) = 2.3799 (autophagosomes). (e) We isolated RNAs from SBMA and control NPC lines (three different NPC lines per human subject) and then measured the expression levels of TFEB target genes including CYCS (cytochrome c), t(4) = 2.844; MCOLN1, t(4) = 3.246; GLA, t(4) = 3.174; ACADM (acyl-CoA dehydrogenase), t(4) = 4.405; COX6A1 (cytochrome oxidase 6A1), t(4) = 5.584; and the TFEB gene itself, t(4) = 0.593. Significant expression reductions were noted in SBMA NPC lines for all tested TFEB target genes. *P 0.05, **P 0.01. Data in d,e are presented as mean ± s.e.m. Individual P values and degrees of freedom are available in the Supplementary Methods Checklist.
CENATAC promotes splicing of A-type minor introns.Figure 5Sashimi plots showing the effect of CENATAC depletion (48hr, HeLaEGFP-AID-CENATAC cells) or ZRSR2 mutations (MDS patient cells) on AT-AC and GT-AG intron retention (left panel, Morc3), and cryptic splice site activation (right panel, Tmem231). CENATAC control represents the parental unedited HeLa cell line. Transcriptome-wide statistics of CENATAC depletion (48hrs) and ZRSR2 mutations on G- and A-type minor intron retention (U12 IR) and cryptic major splice site activation (U2 AS). Only introns showing at least 5 exon-exon junctions reads were included. For U12 IR, a statistical cutoff of Padj <0.05 was used. For U2 AS the probability cutoff of Pr > 0.9 was used. Density plots showing differences in intron retention (ΔΨ) distribution after CENATAC depletion (48 hours) or in samples with ZRSR2 mutations. Hierarchical clustering of A- and G-type intron retention in the unedited parental cell line treated with siGADPH for 48h (column 2), or in the HeLaEGFP-AID-CENATAC cell line treated with siGADPH for 48h (column 3) or with auxin and siCENATAC for 24 or 48 hrs (columns 4 and 5, respectively). Only introns showing a Padj< 0.05 and ΔΨ>0.1 in either the 24hr or 48hr depletion sample were included in the analysis. The A-type and G-type intron terminal dinucleotide subtypes are indicated with different colors in the first column. In case the gene contained multiple introns of the same type, the intron number is indicated in parentheses. Venn-diagram analysis of the MVA patient cell and HeLaEGFP-AID-CENATAC CENATAC depletion datasets. A- and G-type minor introns showing statistically significant intron retention in each dataset were used. See also Dataset EV4. RT-PCR of major (U2-type) AT-AC introns in SYCP2 (intron 5) and TAF2 (intron 1), and minor (U12-type) AT-AC intron in IPO5 (intron 21) on RNA extracted from HeLaEGFP-AID-CENATAC cells depleted of GAPDH or CENATAC. Schematic representations of unspliced/spliced PCR products are depicted on the right. Δ_intron_retention values for the MVA patient cell dataset (compared to the healthy sibling) using all (significant and not significant) minor A-type introns (n=179 for the depletion and n=177 for the MVA patient datasets) and minor G-type introns (n=441 for the depletion and n=446 for the MVA patient datasets). Only introns with on average at least 5 intron-mapping reads were used in the analysis. See also Appendix Figure S12.
Figure 7Kaplan-Meier plot for PFS in patients undergoing immune checkpoint inhibitor therapies stratified by G1 / PD-L1+ tumors (blue) and remaining patients for whom their PD-L1 tumor status is known (red). Same as (A) but including all patients in the study cohort. Remaining patients (red) also included G1 patients with PD-L1 low/negative tumors, G1 patients with unknown PD-L1 tumor status and G2 patients with either PD-L1+ or PD-L1 negative tumors.
Figure 1(a) The phenotypic effects of altering interacting gene partners in SL, DD-SR, and DU-SR interactions. (b) The four inference steps of INCISOR and the datasets analyzed SoF stands for the survival of the fittest). The SR property tested (in red) and rationale (in brown) of each step are also displayed. (c) The resulting DU-SR network (purple nodes denote vulnerable genes and green rescuer genes; the size of nodes is proportional to the number of interactions they have).
Mutations in epg-7 cause defective degradation of SQST-1 aggregates.Figure 1.Mutations in epg-7 cause defective degradation of SQST-1 aggregates. (A and B) In wild-type embryos, SQST-1::GFP is weakly expressed and diffusely localized in the cytoplasm. (A) DIC image of the embryo shown in B. (C) Elevated expression level and accumulation of many SQST-1::GFP aggregates in epg-7 mutant embryos. (D) Compared with wild type and epg-2 mutants, levels of SQST-1 are dramatically elevated in atg-3 and epg-7 mutant embryos in an immunoblotting assay. (E-G) Compared with wild-type embryos (E and F), endogenous SQST-1 shows dramatically elevated levels and accumulates into a large number of aggregates in epg-7 mutant embryos (G). ∼200-cell stage embryos are shown in E-G. (E) DAPI image of the embryo shown in F. (H) Number of SQST-1 aggregates per focal plane in wild type, atg-3 mutants, and epg-7 mutants. (I-L) At the ∼100-cell stage, far fewer SQST-1 aggregates are formed in epg-7 mutants than in atg-3 mutants. (I and K) DAPI images of the embryos shown in J and L, respectively. (M) SQST-1 directly interacts with ZK1053.4 in a GST pull-down assay. (N) Compared with wild-type animals, levels of ZK1053.4::GFP are dramatically elevated in atg-3 and epg-7 mutants. (O-R) ZK1053.4::GFP is weakly expressed in wild-type embryos (O-P) and sqst-1 mutants (R), but forms a large number of aggregates in epg-7 mutants (Q). (O) DIC image of the embryo shown in P. (S) Survival curve of L1 larvae under food depletion conditions in wild type, epg-1 mutants, and epg-7 mutants. This experiment was completed once (wild type: n = 2,964; epg-7: n = 602; epg-1: n = 215). Statistical analysis of L1 survival shows that epg-7 mutants exhibit significant difference to wild type and also to epg-1 mutants (log-rank test, P = 0.000). (T) Loss of function of epg-7 does not ameliorate the degeneration of mec-4::gfp-labeled touch neurons caused by mec-4(u231). (U) Survival curves of wild type and various mutants. sqst-1;epg-7 mutants have a shortened mean and maximum life span. (P = 0.000 for epg-7 or wild type vs. sqst-1;epg-7 in all three repeats; log-rank test). (V) Survival of hydrogen peroxide-treated animals. (In all three repeats, P = 0.000 for epg-7 vs. sqst-1;epg-7; P > 0.2 for wild type vs. sqst-1;epg-7; log-rank test). (W) Survival of animals at the indicated time after heat shock treatment. (P < 0.05 for wild type vs. epg-7 or epg-7 vs. sqst-1;epg-7 at 5, 6, and 7 h). (X) Dauer formation of daf-2(e1370) mutants in different backgrounds.
Figure 2(A) A scheme showing experimental timeline when neuronal inhibitors were added to and removed from the cultured neurons. A cocktail of TTX, D-AP5 and NBQX was added to neurons at DIV 21. (B) Quantitative PCR of Cdc50a during neuronal inactivation and recovery. Neurons without any treatment were used as controls. N = 4 for biological replicates, mean ± SEM, ** p < 0.01. **** p < 0.0001 by two-way ANOVA with Sidak's multiple comparisons test. (C) IHC for CDC50A. CDC50A protein intensities were reduced after neuronal inactivation (day 7), and was recovered after removing neuronal inhibitors (day 13). Scale bar, 10 µm. (D) Relative CDC50A intensities of neurons. N = 5 for biological replicates, mean ± SD, * p < 0.5, **** p < 0.0001 by one-way ANOVA with Holm-Sidak's multiple comparisons test. (E) CDC50A expression at RGC inputs after 5-day consecutive TTX treatment. Scale bar, 10 µm. (F) Quantification of the percentage of CDC50A+/vGlut2+ synapses. N = 5 for biological replicates, mean ± SD, **** p < 0.0001 by Student's test.
Figure 3Immunogold labelling with anti-LBPA antibodies. HeLa MZ cells were treated or not with thioperamide for 18h and processed for electron microscopy. Cryosections were labelled with antibodies against the late endosomal lipid LBPA followed by 5nm protein A-gold. Multivesicular endosomes are pseudocoloured (see Fig EV1 for uncoloured images). Bars, 200nm. The data in (A) were quantified in a double-blind analysis of two sets of 16 micrographs for each condition; the number of gold particles per endosome is shown in a scatter plot (B) for each endosome identified without any bias in each micrograph of the two control (DMSO) and thioperamide-treated (THIO) samples. Distribution of LBPA-containing endosomes in the perinuclear region. Automated unbiased quantification of LBPA fluorescence in the perinuclear region of the cells, after treatment with each compound of the Prestwick library as in Fig 2C. As in Fig 1D, each dot is the average of replicates in the duplicate plates, and ≥600 cells were analysed per compound - only compounds that showed less than 20% toxicity are plotted. The integrated intensity of the LBPA fluorescence signal was measured within a mask of the perinuclear region in each cell, calculated from the nuclei DAPI-staining. The samples treated with thioperamide (pink), trimeprazine (light blue), DMSO (red) and U18666A (green) are indicated. Number of perinuclear LBPA endosomes. The number of individual LBPA-positive structures was measured in the perinuclear region as in (C). The z-factors are shown to evaluate the distribution of endosomes containing LBPA; color code as in (C).