EMBO/sd-character-level-ner huggingface dataset:
Figure 2Bronchial epithelial (BEAS-2B) cells were infected or not with influenza A/Scotland/20/74 (H3N2) virus (IAV) at MOI=1 for 4 h and treated or not with succinate (Suc; 4 mg/mL / 24.7 mM) for 20 h. (a) volcano-plot showing the most significantly regulated canonical pathways determined by microarray analysis compared with mock-treated cells. Each dot represents a specific canonical pathway as determined by GSEA. Pathway representations result from the magnitude of regulation (z-score, x-axis) and significance (−log10 adjusted p-value, y-axis). The dashed horizontal line indicates the statistical significance threshold (p ≤ 0.01 after adjustment with the Bonferroni correction). The two vertical dashed lines show the z-score threshold (-2: repressed; 2: induced). Colored spots characterize the most highly regulated canonical pathways endowed with statistical significance. Data information: Data are the mean ± SEM of independent experiments. Statistical analysis was performed using ANOVA with Holm-Sidak's post-test (a) (*P < 0.01). Bronchial epithelial (BEAS-2B) cells were infected or not with influenza A/Scotland/20/74 (H3N2) virus (IAV) at MOI=1 for 4 h and treated or not with succinate (Suc; 4 mg/mL / 24.7 mM) for 20 h. (b) Representative inflammatory protein-array blots obtained from the supernatants of control- or IAV-infected- or IAV-infected and succinate-treated cells. The table on the right side indicates the 8 most regulated mediators by succinate. Bronchial epithelial (BEAS-2B) cells were infected or not with influenza A/Scotland/20/74 (H3N2) virus (IAV) at MOI=1 for 4 h and treated or not with succinate (Suc; 4 mg/mL / 24.7 mM) for 20 h. (c) Quantification by ELISA of IL-6, IL-8, IP-10 and RANTES in the supernatants of cells infected or not by IAV and treated or not with succinate. Data information: Data are the mean ± SEM of 6 (panel c) independent experiments. Statistical analysis was performed using the Kruskal-Wallis test with Dunn's post-test (c (*P < 0.05) Bronchial epithelial (BEAS-2B) cells were infected or not with influenza A/Scotland/20/74 (H3N2) virus (IAV) at MOI=1 for 4 h and treated or not with succinate (Suc; 4 mg/mL / 24.7 mM) for 20 h. (d, e) Panels show the basal replotted ECAR and OCR (d), and glycolytic capacity (ECAR max) and maximal respiration (OCR max) (e) in cells infected with IAV and treated (green symbols) or not (pink symbols) with succinate. Data were normalized with respect to mock-infected, untreated cells (orange symbols), and were also normalized for DNA content. Data information: Data are the mean ± SEM of d, independent experiments. Statistical analysis was performed using the Kruskal-Wallis test with Dunn's post-test d, e) (*P < 0.05)
Figure 3A Promoter activity of IFN-β or ISRE in SeV-infected 293T cells with or without RBM47 ectopic expression. Cells were infected with SeV for 12 and 24 h, and the promoter activities were determined using the DLR assay.Data are represented as the means ± SD of n = 3 biological replicates. Data information: The data shown are representative of n = 3 independent experiments. NS, non-significant; * P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001 (Student's t-test). B, C Promoter activities of ISRE stimulated by Poly (I: C) (B) or IFN-α (C) in RBM47 overexpressing or knockdown 293T cells. Cells were stimulated with Poly (I: C) (1 μg/mL) or IFN-α (200 U/mL) for the indicated time, and the ISRE promoter activities were determined using the DLR assay. Data are represented as the means ± SD of n = 3 biological replicates. Data information: The data shown are representative of n = 3 independent experiments. NS, non-significant; * P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001 (Student's t-test). D, E mRNA expression of IFIT1, ISG15, and Cig5 in SeV-infected 293T cells overexpressing RBM47 (D) or RBM47 knocked down cells (E). Cells were infected with SeV for 24 h, and gene expression was determined using qRT-PCR.The PCR results are represented as the means ± SD of n = 3 biological replicates. Data information: The data shown are representative of n = 3 independent experiments. NS, non-significant; * P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001 (Student's t-test). F The expression of IFIT1, ISG15, and Cig5 in RBM47-overexpressing or control 293T cells stimulated by IFN-α for 12 h. The PCR results are represented as the means ± SD of n = 3 biological replicates. Data information: The data shown are representative of n = 3 independent experiments. NS, non-significant; * P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001 (Student's t-test). G The expression of IFIT1, ISG15, and Cig5 in WT and RBM47+/- peritoneal macrophages stimulated by IFN-α for 12 h. The PCR results are represented as the means ± SD of n = 3 biological replicates. Data information: The data shown are representative of n = 3 independent experiments. NS, non-significant; * P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001 (Student's t-test).
Figure 2A Relative mRNA expression levels of H2A.B in L1236, L428, HDLM2 HL cell lines and Germinal centre B lymphocytes (GC) compared to the Ramos B lymphocyte cell line. The results are presented as the mean ± SD of three biological replicates. B Western blot analysis of H2A.B protein expression relative to histone H3 protein expression. Error bars represent the standard deviation for three biological replicates, and the red line shows the average. C Subcellular fractionation of L1236 and L428 cells using KCl salt. CN, cytoplasmic and nucleoplasmic fraction, LB, Loosely bound to chromatin, CHR, chromatin fraction. Note that the H3 Western blot signal in CHR is overexposed.
Figure 7A. EU incorporation measured by flow cytometry. Histograms of EU fluorescent signals from two biological replicates for each condition (except no EU). B. SSRP1 staining of the cells in A. C. Volcano plot of changes in transcript counts in 4-OHT-treated cells versus control. Red dots indicate transcripts with fold-change > 2 and FDR < 0.05. n = 2 biological replicates. MA plot showing the difference between the changes in transcript counts in 4-OHT-treated MSCs vs. control and the average expression of the same transcripts between conditions (baseMean). n = 2 cell cultures. E, F. Dot plots with the results of GO enrichment for genes upregulated and downregulated upon FACT depletion.
Macrophage depletion renders tumors more responsive to immune checkpoint blockade therapy.Figure 6PD-L1 expression on MC38 and KPC cells 48 hours following 10Gy irradiation in tissue culture (A,B) or 10 Gy irradiation of tumours (C,D) analysed by flow cytometry. Data presented as mean SEM and analysed by Mann-Witney (n=3, A,B). Data presented as mean SEM and analysed by unpaired t-test (n=5 mice/group, C,D). E-F, Flow cytometric analysis of PD-L1 (E) and PD-L2 (F) on TAMs in MC38 tumours receiving treatment as indicated above. Data presented as mean SEM and analysed by Mann-Witney (n=5 mice/group) G-H, Tumour growth in mice bearing MC38 (G) and KPC (H) tumours receiving the indicated treatments. Data presented for individual mice. Pie charts indicate the number of regressions observed. I-J, Tumour growth in mice bearing KPC tumours mice receiving 10Gy IR + systemic aPD-L1 to the primary lesion (I) ± systemic aPD-L1 + aCSF therapy (J). The difference in tumour volume 8 (I) or 10 (J) days following IR was analysed by unpaired t-test (n=8 mice/group). *P <0.05 **P <0.01 ***P <0.001.
Transcriptional analysis of CT+ and RA+ CMs.Figure 2A-C qPCR of selected transcripts at day 31 to validate (A) enrichment of cardiac markers in GFP+ fractions against GFP− fractions, (B) upregulation of atrial and (C) downregulation of ventricular genes in RA+ compared to CT+ (n = 3).Data information: Data are presented as mean ± SEM. In (A-C), *P < 0.05, **P < 0.01, ***P < 0.001 by unpaired t-test. In (A), for TNNT2, P = 0.0001 for CT− against CT+ and P = 0.0002 for RA− against RA+; for NKX2.5, P = 0.00005 for CT− against CT+ and P = 0.00007 for RA− against RA+. In (B), P = 0.0006 for NPPA and P = 0.0002 for PITX2. In (C), P = 0.02 for HEY2 and P = 0.007 for IRX4. D, E Venn diagram to illustrate overlap of gene lists upregulated (UP) in CT+ CMs (D) and RA+ CMs (E) with genes expressed in atria and ventricles of 15-week-old fetal heart. Red square indicates higher overlap of CT+ with gene list of fetal ventricles or higher overlap of RA+ with gene list of fetal atria. F Pie chart illustrates major classes of gene ontology terms enriched in gene lists upregulated in CT+ and RA+ CMs.
Mitochondrial membrane potential is unchanged in tau-expressing cells.Figure 3A FACS analysis of cells loaded with Rhodamine 123. The geometric mean fluorescence was calculated and expressed as % of the empty vector control. A CCCP-treated cohort was included as a positive control, but not included in the statistical analysis. Data were analysed by one-way ANOVA, F (2, 14) = 0.196, p = 0.8245. B FACS analysis of cells treated with CCCP after 24 h of tau expression. Data were analysed by one-way ANOVA, F (2, 15) = 0.434, p = 0.6555. The DMSO negative control is shown but was not analysed statistically. Data information: Data are given as mean and SEM (n = 5-6 samples/ experimental group from two independent experiments). C Representative dot plots of cells loaded with Rhodamine 123. The gates (black line) were set in unstained cells to distinguish Rhodamine-positive cells (right half) from Rhodamine-negative cells (left half). Data information: Data are given as mean and SEM (n = 5-6 samples/ experimental group from two independent experiments).
Tex11 dosage affects the number of MLH1 foci in meiotic germ cells in both sexes.Figure 4The number of MLH1 foci in pachytene spermatocytes from 3-month-old males positively correlates with increasing Tex11 gene dosage. Quantification of MLH1 foci in pachytene oocytes from the ovaries of Tex11−/−, Tex11+/−, and wild-type embryonic day-17.5 (E17.5) fetuses reveals a positive correlation of Tex11 gene dosage and the number of MLH1 foci.
Figure 4BRCA2-proficient (+BRCA2) or -deficient (-BRCA2) human DLD1 cells were incubated with 1 µM chlorambucil (Chl). Whole cell extracts prepared at the indicated time points during treatment were immunoblotted as shown. GAPDH was used as a loading control. Quantification of chromosome aberrations and chromatid/chromosome break frequencies in BRCA2-proficient (+BRCA2) or -deficient (-BRCA2) human DLD1 cells incubated with 1 μM chlorambucil or 1 µM cisplatin for 72 h. Data were obtained from three independent experiments and normalised to untreated controls. A minimum of 60 Giemsa-stained metaphases were analysed for each sample. Cis, cisplatin; Chl, chlorambucil. Quantification of G2/M cell frequency relative to solvent control, using FACS analyses of cells incubated with 1 μM chlorambucil or 1 µM cisplatin for 48 hours. Cis, cisplatin; Chl, chlorambucil. Human H1299 cells were treated with control (CTR) or indicated siRNAs two days before drugs were added to the media for dose-dependent viability assays. Cell extracts prepared at the time of drug addition were immunoblotted as indicated. SMC1 was used as a loading control. Human H1299 cells were treated with control (CTR) or indicated siRNAs two days before drugs were added to the media for dose-dependent viability assays. Cell extracts prepared at the time of drug addition were immunoblotted as indicated. SMC1 was used as a loading control.
Redistribution of EZH2 leads to misregulation of key developmental regulators A.Figure 2Western-blot analysis of H3K27me3 levels after treatment of cells with an EZH2 inhibitor (EZH2i) or DMSO as control for 12 days. Histone H3 is used as a loading control. Values represent H3-normalized densitometric values of the H3K27me3 bands, and are expressed relative to the relevant DMSO control. UT: untransformed, PN: pre-neoplastic, TR: transformed. Quantification of differentially expressed genes (false discovery rate (FDR) ≤ 0.01, Log2FC ≥ 1/≤-1 and maximal transcripts per million (maxTPM) ≥ 1) detected by RNA-seq in each cellular state upon EZH2i treatment. UT: untransformed, PN: pre-neoplastic, TR: transformed. Venn diagrams showing the overlap between genes upregulated (FDR ≤ 0.01, Log2FC ≥ 1 and maxTPM ≥ 1) by EZH2i treatment in each cellular state. Schematic representation of the multi-step filtering strategy employed to identify functionally important changes in EZH2 distribution induced by neoplastic transformation. Blue and red numbers represent the number of peaks/genes present after each filtering step in untransformed and transformed cells, respectively. UT: untransformed, TR: transformed. Heatmap showing the relative expression of genes identified by the multi-step filtering strategy in the indicated cellular states. UT: untransformed, PN: pre-neoplastic, TR: transformed. Colours represent log2-transformed, row-normalised TPM values.
Figure 4(A) Northern blot analysis of Ifnar1-/- MEFs at 24 h.p.t with the indicated dsRNA or transfection reagent alone (mock) using a probe specific for dsRNA-GFP (top) or dsRNA-RL (bottom). Reactions from a dicing assay using the indicated dsRNA incubated in vitro with immunoprecipitated FLAG-human Dicer were loaded in parallel. Two lanes (delimited by dashed lines) were left empty between the Ifnar1-/- MEFs transfected samples and the in vitro dicing assay reactions. A miRNA marker containing 3 synthetic ssRNA oligonucleotides of 17, 21 and 25-nt in length was run in parallel. Endogenous U6 was used as a loading control. Signals corresponding to the dsRNA-derived siRNAs are indicated with an arrow. The membrane was first probed for dsRNA-GFP (top), then probed for the miRNA marker (top left) and subsequently stripped and reprobed for dsRNA-RL (middle) and finally stripped and reprobed for both U6 (bottom) and for the miRNA marker again (middle left). (B) Western blot of Dicer in d2GFP-expressing Mavs-/- MEFs either not transduced (NT) or transduced to express a non-targeting shRNA control (CTL shRNA) or two different Dicer-specific targeting shRNAs (DICER shRNA 1 and 2). P97 served as loading control. (C) Cells described in (B) were transfected or not (mock) with the indicated Cy5-labeled dsRNAs and d2GFP level in Cy5+cells was monitored by flow cytometry 48 hrs later. Please note that the biphasic pattern of d2GFP level observed in dsRNA-GFP transfected cells is caused by a lower transfection efficiency of dsRNA in these cells giving rise to significant amounts of lowly transfected cells that do not display a decrease level of d2GFP (D) Western blots of Ago2 in parental Mavs-/- MEFs stably expressing d2GFP and three Mavs-/- Ago2-/- clones generated by CRISPR/CAS9-mediated genome engineering. P97 served as loading control. (E) Mavs-/- (parental line) and Mavs-/- Ago2-/- MEFs (clone 5.1) stably expressing d2GFP were transfected or not (mock) with the indicated Cy5-labeled dsRNAs and d2GFP level in Cy5+cells was monitored by flow cytometry 48 hrs later.
Figure 2(A, Nodal-induced gene responses are enhanced further in the absence of Wip1 as analyzed by RT-PCR. Animal caps ere treated with -), no injection of Co MO, Wip1 MO and/or Wip1 mRNA. The dose of reagent injected: Xnr1 (5 pg), Co MO (60 ng), Wip1 MO (60 ng) and Wip1 (1 ng). B) Activin/Nodal-induced gene responses are enhanced further in the absence of Wip1 as analyzed by RT-PCR. Animal caps in (B) were treated with Activin protein (5 ng/ml). (-), no injection of Co MO, Wip1 MO and/or Wip1 mRNA. The dose of reagent injected: Co MO (60 ng), Wip1 MO (60 ng) and Wip1 (1 ng). (C) Knockdown of Wip1 up-regulates the activity of Activin/Nodal-responsive promoter. HEK293T cells transfected with Co siRNA (50 nM) or Wip1 siRNA (50 nM) were stimulated with Activin protein (10 ng/ml) for 10 h and then subjected to luciferase assays. Data are expressed as the mean ± SEM (n=3 biological replicates). ***P<0.001 by unpaired Student's t-test. Depletion of Wip1 promotes epidermal differentiation at the expense of neural fate. (-), no injection of Noggin mRNA. The amount of MO and mRNA injected: Co MO (60 ng), Wip1 MO (60 ng for D, G and J; 30 ng for F and I) and Noggin (5, 10, 20 pg). Scale bar, 150 μm. Depletion of Wip1 promotes epidermal differentiation at the expense of neural fate. Embryos in (E-J) are shown in dorsal views with anterior to the top. The amount of MO and mRNA injected: Co MO (60 ng), Wip1 MO (60 ng for D, G and J; 30 ng for F and I) and Noggin (5, 10, 20 pg). Scale bar, 150 μm. (K, Activin induction of p21 is up-regulated upon Wip1 knockdown. HEK293T cells were transfected with Co siRNA or Wip1 siRNA and 48 h after siRNA transfection, were stimulated with Activin (10 ng/ml) for 3 to 12 h as indicated t=0 in (K) is 48 h after siRNA transfection. P-Smad2 serves as an indicator of active Activin/Nodal signaling. Arrows ndicate non-specific bands. L) TGF-β induction of p21 is up-regulated upon Wip1 knockdown. HEK293T cells were transfected with Co siRNA or Wip1 siRNA and 48 h after siRNA transfection, were stimulated with increasing concentration of TGF-β1 (10, 20 ng/ml) for 3 h.
PI5P4K2A and PI5P4K2C Regulate Accumulation of Mutant Huntingtin Exon 1 (A and B) Western blot analysis of LC3-II and tubulin levels and quantification of LC3-II/tubulin ratio in SKNSH and MEFs treated for 2 hr with carrier alone or in combination with PI(5)P di-C16 (10 μM) in the absence and presence of BAF (mean ± SEM).Figure 4(A and B) Western blot analysis of LC3-II and tubulin levels and quantification of LC3-II/tubulin ratio in SKNSH and MEFs treated for 2 hr with carrier alone or in combination with PI(5) P di-C16 (10 μM) in the absence and presence of BAF (mean ± SEM). (C) HeLa cells treated with PI5P4K2A or PI5P4K2C siRNA for 5 days or with pCMV-PI5P4K2C overexpression construct for 48 hr were transfected with EGFP-httQ74 for the last 48 hr. Percentage of cells with EGFP-positive aggregates is shown in the graph; n = 500 cells (mean ± SEM). Cells expressing control vector typically have 25% of aggregates and we set the control at 100% to enable comparison and statistics from independent experiments. (D and E) Percentage of cells with EGFP-positive aggregates was scored in HEK293 cells treated with PI5P4K2C siRNA for 5 days and cotransfected with EGFP-httQ74 for the last 48 hr (D), or in COS7 and SKNSH cells (E), transfected with PI5P4K2C plasmid together with EGFP-httQ74 for the last 48 hr. (F and G) WT (Atg5+/+) or autophagy-deficient cells lacking the key autophagy gene Atg5 (Atg5−/−) MEFs were treated with PI5P4K2C siRNA (5 days) or PI5P4K2C plasmid (48 hr) together with EGFP-httQ74 for 48 hr. The percentages of transfected cells with EGFP-httQ74 aggregates were assessed and shown in the graph; n = 500 cells (mean ± SEM). Representative confocal images are shown in (G). See also Figures S3 and S4.
Effects of knockdown of cV subunits in combination with other treatments.Figure 4A‐D Relative mtDNA level (A, B) and MitoSox fluorescence (C, D) following 5 days of treatment with dsRNA against the genes indicated. E‐G Relative mtDNA level (E), MitoSox fluorescence (F) and TMRM fluorescence (G) of cells stably expressing AOX or empty vector, as indicated, after 5 days of treatment with dsRNA against CG2968, as shown. Note that these cells were cultured in the presence of hygromycin to maintain the AOX‐expressing or control plasmid. H Indicated parameters, following 5 days of treatment with dsRNA against CG2968, in cells grown in galactose‐containing (Gal) medium.
Figure 5A Experimental scheme. In vitro HMTase assays. Recombinant H3N-GST (WT or K9R) pre-ubiquitylated by the CLRC was purified and used in the HMTase assay with 6×His-tagged recombinant Clr4 (His-Clr4). Proteins were analyzed by SDS-PAGE and Coomassie Brilliant Blue (CBB) staining (left) and autoradiography (right). C,D In vitro HMTase assay using disulfide-linked ubiquitylated histone H3. H3K14CUb and control histone H3 (H3C110A) were used as substrates in the HMTase assay, and analyzed The 3H-methyl group incorporation was measured using a liquid scintillation counter (D). Error bars indicate the standard deviations calculated from three independent experiments.
Figure 7A, B SAXS profiles of the H1-nuclesomal arrays in 0 (TE), 1, 2.5 mM MgCl2 (A) and 5, 10 mM MgCl2 (B) are shown as plots of log(I × S2) vs. 1/S (I, intensity; S, scattering vector (1/nm)). C, D SAXS profiles of the native chicken chromatin in 0 (TE), 0.5, 1 mM MgCl2 (C) and 2.5, 5 mM MgCl2 (D) are shown as plots of log(I × S2) vs. 1/S (I, intensity; S, scattering vector (1/nm)). E The "in silico oligomer" models were constructed in environments containing 100, 50, and 25 randomly and tightly packed 12-mer 30-nm fiber models (Fig. 3E). The nucleosome concentration was 0.5 mM. The 100-fiber model was drawn using MolScript (Kraulis, 1991). The broken-lined squares show magnified regions. F The modeled scattering profiles, yielding average among-model values, have prominent peaks at ~30-40, 11, and 6 nm. This shows that the in silico oligomers retain characteristics of 30-nm fibers. Note that the modeled scattering profile is very distinct from the SAXS profiles in Figs. 3B, 7B and D, and also 8B (center and right).
Figure 4A: Percentage of methylated CpG dinucleotides profiled across the Esrrb and Nanog enhancer and the Nanog promoter in sorted SSEA-1+ / EsrrbHi, EsrrbMed or EsrrbNeg E-GFPd1 ESCs and TNG E-2a-tdT EpiSC. CpG methylation was assessed by measuring protection from digestion of the HpaII, AciI, Hin6I or TaqI restriction sites (indicated by vertical lines in the gene structure maps derived from the mouse reference genome (mm9) and expressed relative to the TSS in ESCs Values represent total CpG methylation levels (5mC + 5hmC). Error bars: standard deviation of the measures in four independent experiments. B: Methylated CpG dinucleotides across the Esrrb enhancer were assessed SSEA-1+ / EsrrbHi E-GFPd1 ESCs were sorted and placed back in culture overnight before resorting and methylation analysis relative to Esrrb- E-GFPd1 ESCs. Error bars: standard deviation of the measures in three independent experiments. C: The sorted E-GFPd1 ESCs populations were assessed by immunoblot for Esrrb and Nanog (left) or Oct4 (right) by fluorescence-based detection (Licor). D: Quantitative ChIP-PCR analysis of OCT4, NANOG and ESRRB binding and histone modifications at the Esrrb and Nanog enhancers and the Nanog promoter in the sorted populations Error bars: standard deviation of the measures in two (NANOG, ESRRB, H3K27ac) or three (OCT4, H3K4me3, H3K4me1) independent ChIP experiments, each performed on pooled chromatin from at least three independently sorted samples. The diagrams at the bottom show the approximate position of the regions analysed for transcription factor binding or histone modifications (in color or black), along with the relative control genomic locations (grey).
Effects of HCV nonstructural proteins on the induction of Rubicon and autophagosomes.Fig 7(A) Western-blot analysis of Huh7 cells transfected with the expression plasmids of HA-tagged GST and various HCV nonstructural proteins. Cells were lysed 48 hours after transfection for analysis. The localizations of molecular weight markers are indicated. The asterisk denotes a nonspecific protein band. (B) Analysis of GFP-LC3 puncta in stable Huh7 cells that expressed GFP-LC3. Cells were transfected with various expression plasmids for 48 hours and immunostained with the anti-HA antibody (red color). GFP-LC3 puncta were apparent in cells that expressed HCV NS4B. (C) The average number of GFP-LC3 puncta per cell shown in (B). The results represent the mean of >30 cells.