EMBO/sd-character-level-ner huggingface dataset:
Figure 2Immunofluorescent analysis of TDP-43 in control and mutant TDP-43 iPSC-derived motor neurons. Upper images represent control and lower mutant TDP-43 motor neurons. Presented immunofluorescent images of control (C2) and mutant TDP-43 (P2), Scale bar: 20µm. Corresponding profile intensity plot of panel A. Quantification of the nucleo-cytoplasmic ratio (N/C) of TDP-43 fluorescent intensity of pooled control and pooled mutant motor neurons, unpaired Mann-Whitney-test. Western blot analysis of cytoplasmic fraction with a positive nuclear control (C) confirming the fractionation purity, quantification in E, ratio paired-t-test. Western blot analysis of nuclear fraction with a positive cytoplasmic control (C) confirming the fractionation purity, quantification in G, ratio paired-t-test. Immunofluorescent analysis showing fluorescence intensity of phosphorylated TDP-43 (pTDP-43). Scale bar: 20µm. Presented immunofluorescent images of control (C2) and mutant TDP-43 (P2). I represents its corresponding profile intensity plot of the selected (square) region. Scale bar: 10µm, J quantification pooled control (C0=10, C1=10 and C2=10) n=30 and pooled mutant TDP-43 (P1=10, P2=10 and P3=10) n=30 cells from each differentiation. Unpaired Mann-Whitney-test. Western blot analysis showing pTDP-43, quantification in L, ratio paired-t-test.
induces glucose metabolism to aerobic glycolysis reprogramming in NPC cells.Figure 4A, Gene expression profiles from CNE2 cells overexpressed with INSL5 or the vector control. The genes are shaded with green, black or red in the heat map to indicate low, intermediate or high expression, respectively. B, Functional annotation clustering of genes regulated by INSL5 in CNE2 cells is shown. Significantly enriched groups nominated by the gene ontology term are ranked based on the group enrichment scores. Red: biological process. Blue: signaling pathway. C, The signal pathway of the subset of genes involved in metabolism was assessed by Gene Set Enrichment Analysis. D, Analysis of glycolytic gene expression by immunoblot in INSL5 overexpressed or knock-down cell lines. E, Metabolites were purified from five (CNE1 cell line and HK1 cell line) or seven (CNE2 cell line) independent samples of two vector and INSL5 overexpressed cell lines and analyzed by liquid chromatography-mass spectrometry (LC-MS). Red: glycolytic intermediate metabolites. Green: tricarboxylic acid cycle (TCA cycle) intermediate metabolites. PPP: pentose phosphate pathway. The color scale bar represents Z-score of the metabolites' level. F, The extracellular acidification rate (ECAR) was measured in cells with or without INSL5 overexpression using a Seahorse XF96 Extracellular Flux analyzer. Glu, glucose; Oligo, oligomycin. G, Statistical analysis of the effects of INSL5 overexpression on glycolytic activity. H, CNE1-vector and CNE1-INSL5 cells were grown in the presence of the fluorescent glucose analog 2-NBDG (Invitrogen) for 15min, and glucose uptake was then quantified using flow cytometry (FACS) (left panel). Statistical analysis of the glucose uptake was showed in the right panel. glucose consumption and lactate production (I) in CNE1-vector and CNE1-INSL5 cells. basic ATP level or the ATP level of cell which was treated by 1uM oligomycin or 50uM 2-DG for 24h (J) in CNE1-vector and CNE1-INSL5 cells. HK2 enzyme activity (K) in CNE1-vector and CNE1-INSL5 cells. L, ATP concentration, HK2 enzyme activity and lactate production in INSL5 wide-type or knock down HNE1-EBV cells.
O-GlcNAc regulation of STAT1 in IECs.Figure 7 A Expression of phosphorylated and total STAT3 and NF-κB p65 in the colon of wildtype and Vil-Ogt KO mice  B Ileum and colon IECs from 10-week-old wildtype and Vil-Ogt KO mice were isolated for RNA-sequencing analyses. The heatmaps of the sample-to-sample distances are shown  C Volcano plots of gene expression changes. The x-axis specifies the fold-changes (FC) and the y-axis specifies the negative logarithm to the base 10 of the p-values adjusted for multiple testing with the Benjamini-Hochberg procedure. FC > 2 and p < 0.05 genes are shown in solid dots  D Most significantly enriched canonical signaling pathways in differentially expressed genes, determined by the Comparison Analysis module in the IPA program  E Top candidate transcription factors controlling the observed differentially expressed genes, predicted by DiRE  F Levels of STAT1 protein in IECs determined by immunoblotting and O-GlcNAc modification of STAT1 determined by immunoprecipitation using the O-GlcNAc antibody followed by Immunoblotting   G, H Caco-2 cells were treated with 100 uM Ac45S-GlcNAc for 24 hours, levels of STAT1 and global O-GlcNAcylatio were determined  G, H Caco-2 cells were treated with 100 uM Ac45S-GlcNAc for 24 hours, levels of STAT1 an STAT1 O-GlcNAcylation (H) were determined  I siRNAs against scrambled sequence or human OGT were transfected in 293T cells and STAT1 levels were determined 48 hours later   J The pGAS-luc Cis-Reporter plasmid was co-transfected with siRNAs in 293T cells for luciferase assay to determine the transcriptional activity of STAT1 (n = 4, P=0.0003)
Figure 5Cartoon illustrating the recognition of Nek2A by APC/CMCC. KEN box and D box dependent substrates such as securin and cyclin B do bind APC/CMCC. In contrast, cyclin A and Nek2A bind APC/CMCC-open. Cyclin A is ubiquitinated by UbcH10 and Nek2A has more chances of being ubiquitinated once bound to the APC/CMCC-open by using both UbcH10 and UbcH5. The positioning of UbcH5 with respect to the APC/C catalytic site is based on mutagenesis data
Effects of KSR and WNT3A on PGC‐precursor and PGCLC inductionEffects of KSR and WNT3A during PGC‐precursor induction on the expression of selected pluripotency, PGC, and a mesodermal gene of d2 cultures.Figure 4Effects of KSR and WNT3A during PGC‐precursor induction on the expression of selected pluripotency, PGC, and a mesodermal gene of d2 cultures. The value for iPSCs is set as 1, and values are on log2 scale. Data are presented as means ± SD (n = 3). Effects of KSR and WNT3A on the induction of d6 PGCLCs as analyzed by FACS gated for TRA‐1‐81 and cKIT. Gene expression analysis of selected PGC and hematopoietic markers in d6 PGCLCs that were cultured in 0% (control) or 20% KSR (KSR) condition during PGC‐precursor induction. Samples were calibrated with iPSC values, and iPSC values depict 1. Data are presented as means ± SD (n = 3). Immunofluorescence analysis for OCT4 (red), BLIMP1 (green), and T (yellow) in d2 precursors cultured in increasing KSR concentrations. Nuclei were stained with Hoechst (blue). Scale bar: 100 μm.
a) Cell death was analysed after the activation of death pathways in the presence and absence of enforced Atg5 expression (18 h cultures).figf1(a) Cell death was analysed after the activation of death pathways in the presence and absence of enforced Atg5 expression (18 h cultures). Values are means ± s.e.m. of three independent experiments. (b) The cell death regulated by Atg5 is apoptosis. All data presented are from 5 h cultures. Caspase-3-like activity analysed in HeLa cells (graph). Morphology of Jurkat cells is also shown. The numbers in the lower right corner indicate the percentage of cell death, which was determined in parallel experiments using flow cytometry c) Staurosporine induced redistribution of phosphatidylserine was enhanced in Atg5 overexpressing Jurkat cells and blocked by z-VAD-fmk. Numbers indicate the results of the quantitative analysis (percentage) of each dot blot. d) Reduction of drug-induced cell death by silencing of Atg5. Values are means ± s.e.m. of three independent experiments (18 h cultures). The effects of the Atg5 siRNA on Atg5 and GAPDH levels were analysed by immunoblotting. The asterisks indicate P 0.05. n.s., no significant difference.
EC mitigates sepsis-associated inflammation in vivo.Figure 5A C57BL/6mice were treated (i.v.) with 500 μg EC or DPPC control 2 h prior to i.p injection of 150 ng/g LPS together with 800 μg/g D-galactosamine. 4 h after LPS application, lungs were perfused with PBS and embedded in paraffin. Tissue sections were hematoxylin-stained to visualize adherent cells. Bars represent 100 µm.B, C Leukocyte adhesion to lung microvascular endothelium as determined by morphometric image analysis of lung tissue sections is presented for individual vessels in (B) and as averages of single mice in (C). Pooled data of two independent experiments are shown (n = 10 for EC, n = 14 for DPPC). Unpaired two-tailed t-test. D, E C57BL/6mice were treated with EC or DPPC by intra-tracheal instillation at 18 h (50 μg) and 1.5 h (100 μg) prior to i.p. injection of 150 ng/g LPS and 800 μg/g D-galactosamine. Bar graphs represent absolute numbers of total infiltrating cells and of neutrophils (D). Unpaired two-tailed t-test. Data represent mean ± SEM from one of two independent experiments with at least 6 mice per group. (E) Dot plots depict exemplary gating of lungneutrophils on pregated CD45+CD11c−SiglecF− BAL cells of EC-/LPS-treated and DPPC-/LPS-treated mice. F, G The concentrations of IL-6 (F) and IL-12 (G) in the BAL of mice treated as in (D) were quantified by ELISA. Data (mean ± SEM) are representative of two independent experiments with 3 mice per group. ****P < 0.0001; unpaired two-tailed t-test. C57BL/6mice were treated with EC or DPPC by intra-tracheal instillation at 18 h (50 μg) and 1.5 h (100 μg) prior to i.p. injection of 150 ng/g LPS and 800 μg/g D-galactosamine. (E) Dot plots depict exemplary gating of lungneutrophils on pregated CD45+CD11c−SiglecF− BAL cells of EC-/LPS-treated and DPPC-/LPS-treated mice.
Figure 2A BAK-6H on liposomes was activated or not with cBID for 60 mins prior to deuteration for 10s, 60s, 600s, or 3600s. Incorporation of deuterium was assessed by mass spectrometry. Change in deuteration at each time point is expressed relative to BAK on liposomes without cBID. B Deuteration of BAK in a membrane compared to inactive BAK is mapped onto the structure of inactive monomeric BAK C Deuteration of BAK in a membrane compared to inactive BAK is mapped onto the structure of an activated BAK BH3:groove dimer
Figure 3(A, B) Immunofluorescence analysis of the old APPPS1 slice in co-culture (14 DIV) using antibodies 6E10 (green) and CD68 (red). Nuclei were counterstained using DAPI (blue). Image of boxed region in A is depicted at higher magnification in B and reveals clustering of several CD68 positive cells around amyloid plaques. Scale bars: 100 µm (A) and 10 µm (B).(C) Quantitative analysis of plaque-associated CD68 positive cells in co-culture samples at 7 and 14 DIV reveals a trend towards increased number of CD68 positive cells. The values represent mean ± SEM from at least 3 independent experiments, including total of at least 6 independent slice culture dishes (n.s.= not significant).(D) Quantitative analysis of co-culture samples at 7 and 14 DIV reveals a decrease in plaque size. The values represent mean ± SEM from at least 3 independent experiments, including total of at least 6 replicates (***P < 0.001).(E) Quantitative analysis of plaques surrounded by CD68 positive cells (clustered plaques) in co-culture samples at 7 and 14 DIV reveals an increase in the number of clustered plaques. The values are expressed as percentages of clustered plaques from the total number of amyloid plaques. The values represent mean ± SEM from at least 3 independent experiments, including total of at least 6 independent slice culture dishes (***P < 0.001). (F) Immunofluorescence analysis of the old APPPS1 slice in co-culture (14 DIV) treated with cytochalasin D (CytoD) and vehicle control (Ctr) and immunostained with M3.2 (green) and CD68 (red). Inhibition of phagocytosis by CytoD blocks amyloid plaque clearance. Scale bar: 50 µm.(G) Quantitative analysis of core-only plaques in the old APPPS1 slice in co-culture (14 DIV) treated with CytoD and Ctr reveals a decreased number of core-only plaques upon CytoD treatment. The values are expressed as percentages of core-only plaques from the total number of amyloid plaques. The values represent mean ± SEM from 3 independent experiments, including total of 6 independent slice culture dishes (***P < 0.001).
Rpd3 represses transcription, H2B ubiquitination and H3K79 methylation at its target sites.Figure 2ChIP-seq and RNA-seq data for genes ranked on H3K79me3/H3 in rpd3Δ/WT, smoothed using locally weighed regression. The gray band around the line shows the 95% confidence interval. Vertical dashed lines separate 4 groups with distinct changes upon RPD3 deletion. ChIP-seq data of H3K79me1, H3K79me3 and H2Bub were generated in this study (plotted is the average coverage in reads per genomic content, RPGC), Rpd3 binding, H4ac and WT gene expression data were from (McKnight et al, 2015), and the relative expression in rpd3Δ/WT from Kemmeren et al (2014). H3K79me3/H3 ChIP-qPCR efficiencies (relative to a non-transcribed region, which was unaffected by RPD3 deletion) in wild-type and in rpd3Δ cells harboring empty or RPD3-encoding CEN plasmids. The H188A and H150A-H151A mutations have previously been shown to abrogate catalytic activity (Kadosh & Struhl, 1998). Error bars indicate standard deviation of three biological replicates. p<0.05 (*), p<0.01 (**) and p<0.001 (***) by two-way ANOVA, comparison to wild type. Gene set enrichment analysis on genes ranked on H3K79me3/H3 in rpd3Δ/WT; all genes have been ranked and the ranks of the genes in the subsets are indicated by vertical lines. Meiotic (C) and Ume6-bound (D) genes are enriched among the genes at which Rpd3 represses H3K79 methylation, and subtelomeric genes (<30 kb of telomere) (E) are enriched among genes at which H3K79 methylation is decreased in rpd3Δ cells.
Figure 4A 3T9 fibroblasts expressing the indicated MycER proteins and control cells (EV) were treated with OHT (4h) and subjected to biochemical fractionation in three independent experiments. The quantity of MycER protein in the different fractions was quantified by western blotting (shown in Fig. EV4A) and the relative proportion in the various fractions plotted as average and standard deviation. B Single molecule tracking at high frame rate: the time of ttl = 10ms between two images allows to estimate the distribution of displacements, that is then fit by a three-component diffusion model to estimate the fraction of molecules immobilized on chromatin. Inset: Average bound fraction (see Fig. EV4C and Methods). Note that MycRA displays a significantly lower bound fraction than MycWT and MycHEA (ncells= 34, 30 and 35, and ndisplacements= 78550, 59783 and 83801 for MycWT, MycRA and MycHEA, respectively). Error-bar: SD. Statistical significance evaluated by permutation tests Data information: , the statistical significances are indicated relative to the WT control (or RA vs. HEA, as indicated): * P < 10-4; # P < 10-3; + P = 3.1x10-3; ns not significant C Single molecule tracking at lower frame rate (ttlspanning between 200 ms and 2s) allows to quantify the distribution of residence times (i. e. the duration of binding events). Inset: Average residence times (see Fig. EV4E-F and Methods). The data reveal a significantly shorter average for MycRA, relative to either MycWT or MycHEA (ncells= 35, 35 and 31, and nBound − molecules= 2452, 2084 and 2171 for MycWT, MycRA and MycHEA, respectively). Error-Bar: SD. Statistical test: Anova-Tukey Data information: the statistical significances are indicated relative to the WT control (or RA vs. HEA, as indicated): * P < 10-4; # P < 10-3; + P = 3.1x10-3; ns not significant
Fig.Figure 7LU-NB-3 PDX cells were treated with 50 nM of IBL-302 for 48 hours and subsequently analyzed by RNAseq and mass spectrometry. A-C. Heat map of significantly differentially expressed genes across treatment groups from RNAseq data (FDR<0.1) (A). Genes defined as differentially expressed that were also present in MS and phospho-MS are visualized in (B) and (C), respectively. LU-NB-3 PDX cells were treated with 50 nM of IBL-302 for 48 hours and subsequently analyzed by RNAseq and mass spectrometry. D. Gene ontology generated from significantly altered genes from RNAseq data. LU-NB-3 PDX cells were treated with 50 nM of IBL-302 for 48 hours and subsequently analyzed by RNAseq and mass spectrometry. E. Gene ontology generated from significantly altered proteins from mass spectrometry proteome data. LU-NB-3 PDX cells were treated with 50 nM of IBL-302 for 48 hours and subsequently analyzed by RNAseq and mass spectrometry. F. Gene Ontology generated from significantly altered phospho-proteins from mass spectrometry phospho-proteome data. LU-NB-3 PDX cells were treated with 50 nM of IBL-302 for 48 hours and subsequently analyzed by RNAseq and mass spectrometry. G. Cross-platform sample correlations of RNAseq and mass spectrometry proteome data. Pearson's correlation coefficients are displayed.
DDR activation mediates transcriptional upregulation of ACE2 gene.Figure 4A Gene set enrichment analysis showing enriched pathways from the MSigDB_Hallmark gene-set library. Bars are colored according to p value, with significantly enriched pathways (P < 0.05) in blue. Combined score: combined value of both the p-value and z-score. B Relative luciferase activity in HeLa shTRF2 following ionizing radiation (5 Gy) or TRF2 knockdown upon doxycycline-induced shTRF2 expression (n=3 independent experiments). Error bars represent the s.e.m. ﻿*P<0.05. Two-way paired ANOVA.
Figure 2(A-C) The relative levels of (A) canalicular membrane (BA) transporter Bsep, Mrp2, Mdr1b, Mdr2, Abcg5 and Abcg8 mRNA transcripts, (B) basolateral membrane BA transporter Mrp3, Mrp4, Ostβ, Ntcp and Oatp1a1 mRNA transcripts, and (C) BA synthetic enzyme Cyp7a1, Cyp7b1, Cyp8b1, Cyp27a1, and Cyp2c70 mRNA transcripts. Wild type, wild type mice, n=6; Heterozygote, Sema7aR145W heterozygous mice, n=5; Homozygote, Sema7aR145W homozygous mice, n=7. Data are shown as means ± SD. *P < 0.05 versus the WT mice, #P < 0.05 versus the heterozygous mutant mice. The data were analyzed by the independent-samples Student's t-test and the Mann-Whitney U test. (D) Western blot analysis of the relative levels of Bsep, Mrp2, Mdr2, Mrp3, Mrp4, Ostα/β, Ntcp, Cyp7a1 and Cyp8b1 protein expression in mouse livers. Contrary to the mRNA alterations, the levels of canalicular membrane Bsep and Mrp2 proteins were dramatically reduced in homozygous mouse livers, suggesting that the decreased these proteins were might be mediated by post-translational regulation in the livers of Sema7aR145W mutant mice. Wild type, wild type mice, n=4; Heterozygote, Sema7aR145W heterozygous mice, n=5; Homozygote, Sema7aR145W homozygous mice, n=5. *P < 0.05 versus the WT mice, #P < 0.05 versus the heterozygous mutant mice. The data were analyzed by the independent-samples Student's t-test and the Mann-Whitney U test. (E, F) Multiplex IF analyses of Bsep and Mrp2 expression in the livers (E) and primary hepatocytes in sandwich cultures (F) from WT and homozygous mice. Scale bar, 50 µm. (G) Multiplex IF analyses revealed that transfection with the SEMA7A_WT or SEMA7A_R148W construct decreased the levels of canalicular membrane Bsep and Mrp2 expression in mouse primary hepatocytes in collagen sandwich cultures in a dose-dependent manner (0, 0.2, 1.0 μg per well of 6-well plate; 1.5 ml culture medium per well). Scale bar, 50 µm. Together, the Sema7aR145W mutation can reduce hepatic Bsep and Mrp2 protein expression and lead to intrahepatic BA accumulation and cholestasis (Appendix Table S16). Subsequently, cholestasis triggered an adaptive response in the liver by down-regulating the expression of BA synthetic enzymes of Cyp7a1 and Cyp8b1 and up-regulating the expression of BA efflux transporters Mrp3, Mrp4 and Ostα/β.
Figure 5Direct regulation between genes is defined based on causal attribution analysis and indirect regulation of an induced gene is defined if a gene is differentially expressed regardless of whether attribution analysis indicated a direct regulatory relationship. A. Edges between both genes with induction experiments and predicted regulators were formed based on regulatory interactions predicted from individual experiments (as shown in Figure 3C). For this visualization, major regulators selected as per Figure 3C are rooted to the induced TF regardless of whether they were directly or indirectly regulated by this gene. Predicted regulators are linked to GO categories based on having a significant overlap with their predicted targets. Similarly, genes with an induction experiment are linked to GO categories based on overlap of either direct or indirect targets with GO categories. Validated nodes (green) are genes where validation experiments confirmed a significant overlap between measured gene-regulator connections and model-predicted coefficients. Invalidated nodes (red) are genes where validation experiments failed to confirm model-predicted coefficients. B. Local networks based on upstream direct/indirect regulators and downstream direct targets of three validated regulators.
Macrophages infected with Mtb-Δeis show reduced cell viability and increased caspase-independent cell death.Figure 4(A) BMDMs were infected with Mtb-WT, Mtb-Δeis, or Mtb-c-eis (MOI = 10) for the indicated periods of time, washed to remove unbound mycobacteria, and then incubated in complete DMEM at 37°C in 5% CO2. Cell viability was assessed by PI staining and then examined by fluorescence microscopy. (B and C) Experimental conditions were identical to those outlined in panel A. BMDMs were infected with the three strains of mycobacteria for 36 h. B) Apoptosis was assessed using a TUNEL/apoptosis detection kit, according to the manufacturer's protocol. Cells were then examined under a laser-scanning confocal microscope (LSM 510; Zeiss). Percentages of TUNEL-positive, PI-positive, and TUNEL-/PI-double-positive cells were calculated. Data are representative of three separate experiments. (C) Morphological changes in BMDMs infected with Mtb-WT, Mtb-Δeis, or Mtb-c-eis at MOIs of 1 and 10. Representative images are shown. Scale bars: 50 µm (low magnification), 20 µm (high magnification). (D) Macrophages were infected with Mtb-WT, Mtb-Δeis, or Mtb-c-eis in the presence or absence of zVAD-fmk (20 µM) or 3-MA (10 mM). Staurosporine (STS; 500 nM) was used as a positive control. After 36 h, cells were stained with PI and then examined by fluorescence microscopy. Data are presented as the mean±SD of at least three separate experiments, each performed in duplicate. *p<0.05, ***p<0.001, vs. Mtb-WT-infected condition (A and B); Mtb-Δeis-infected condition without inhibitors (D). UI, uninfected.
Figure 6CD4+ T cells were isolated by negative selection from WT, EGR4-/- mice. (A) WT or EGR4-/- cells were loaded with Fura-2AM and plated on anti-CD3/CD28 antibodies for 2 hrs. Senicapoc (4 μM) or Tram-34 (5 μM) were added where marked by the arrow. CD4+ T cells were isolated by negative selection from WT, EGR4-/- mice. (B) CD4+ T cells were isolated by negative selection from WT or EGR4-/- mice and incubated with anti-CD3/CD28 antibodies for 6 hrs in the presence or absence of either senicapoc, TRAM-34 or Shk-Dap22 before measuring expression of IFNγ levels by FACS analysis. Data are mean±SEM; 3 biological replicates were examined; each biological replicate includes 2 technical replicates. *** p < 0.001; **** p < 0.0001. CD4+ T cells were isolated by negative selection from WT, EGR4-/- mice. (C) To measure the effect of KCa3.1 inhibition of cell proliferation, WT and EGR4-/- CD4+ T cells were isolated from the spleen by cell sorting and stained with CFSE. Cells were incubated with anti-TCRβ antibodies in the presence of vehicle, Senicapoc (5 µM) or TRAM-34 (5 µM) for 4 days and then collected for FACS analysis. (D) Generation analysis was completed for all data using FlowJo software. Data are mean±SEM; 3 biological replicates were examined; each biological replicate includes 2 technical replicates.