EMBO/sd-character-level-ner huggingface dataset:
Overexpression of DRP1/Fis1 and Bnip3 proteins induces changes in the mitochondrial network in vivo.Figure 4Overexpression of DRP1/Fis1 and Bnip3 proteins induces changes in the mitochondrial network in vivo. In vivo imaging of the mitochondrial network was performed in muscles expressing Bnip3, an atrophy‐related gene under FoxO3 control, and DRP1/Fis1 proteins. Adult muscles were transfected with plasmids encoding mitochondrially targeted yellow fluorescent protein (mtM13‐YFP) and either mock vector, Bnip3, HA‐DRP1, Fis1 or DRP1/Fis1. Seven days later (for Bnip3) or 12 days later (for DRP1 and Fis1), muscles were observed in vivo with confocal microscopy. TMRM was injected into the muscles to monitor mitochondria, which retain membrane potential. Muscles transfected with mtM13‐YFP and mock vector showed the orderly pattern of intermyofibrillar and subsarcolemmal mitochondria (upper panel‐IMF). Scale bar: 20 μm.
N histone binding activity preserves nucleosome integrity during transcription.Figure 4(A) Samples from the experiment described in Figure 3A were also processed for MNase-Seq. For each of the three indicated strains, the histograms represent the normalized DNA sequence read count per million reads (RPM), across a sample gene body. (B) Average of the normalised read density (RPM) for the MNase-Seq data from two independent experiments, for regions spanning 1kb upstream and downstream of the Transcription Start Site (TSS) of actively transcribed genes across the yeast genome (as above, the analysis focussed on 821 active genes, as described in Materials and Methods). (C) Samples from the same experiment were further processed for ChIP-Seq using antibodies to histone H3K4me3. For each of the three indicated strains, the normalised read count per million reads is presented across a sample gene body. (D) Average of the normalised read density (RPM) for the H3K4me3 ChIP-Seq datasets from two independent experiments, for regions spanning 1kb upstream and downstream of the Transcription Start Site (TSS) of actively transcribed genes. (E) Principal component analysis of two independent experiments, for the H3K4me3 ChIP-Seq signals across the yeast genome in the three indicated strains. (F) Ratio of the H3K4me3 ChIP-Seq signal (TSS to +500 bp) in cells expressing Spt5-3A and cells expressing wild type Spt5. The boxes extend from the 25th to 75th percentiles whilst the 'whiskers' illustrate the minimum and maximal values and the lines within each box correspond to the median values. The data are expressed as a function of the gene expression level, based on the read density of the ChIP-Seq data for CTD-Ser5P (from TSS to TTS) in Figure 3, ordered from lowest to highest values in four quartiles (colour coded as indicated). The correlation between H3K4me3 in cells expressing Spt5-3A (spt5-AID GAL-SPT5-3A) and control cells (spt5-AID GAL-SPT5) is progressively worse from Q1 to Q4, for genes expressed at progressively higher levels.
Fig.Figure 6Representative confocal z-stack images of LV-infected dendritic segments in the DG granule neurons from control and susceptible mice with different treatment. White arrows indicate stubby spines, yellow arrows indicate mushroom spines, and blue arrows indicate thin spines. B-E Quantification of total (B), stubby (C), mushroom (D) and thin (E) spine density in DG from control and susceptible mice infected with LV-GFP or LV-siCD39 (n = 15 segments from 4 mice/group. For total density: Treatment F2, 42 = 3.12, P = 0.0544; Ctrl - LV-GFP vs CSDS - LV-siCD39, P = 0.0312; CSDS - LV-siCD39 vs CSDS - LV-GFP, P = 0.0425; stubby density: Treatment F2, 42 = 10.93, P=0.0002; P = 0.0026; CSDS - LV-CD39 vs CSDS - LV-GFP, P < 0.0001; mushroom density: Treatment F2, 42 = 0.34, P = 0.7161; thin density: F2, 42 = 1.00, P = 0.3758. One-way ANOVA with Fisher's LSD test). F Representative confocal z-stack images of LV-infected dendritic segments in the DG granule neurons from control and susceptible mice with different treatment. White arrows indicate stubby spines, yellow arrows indicate mushroom spines, and blue arrows indicate thin spines. G-J Quantification of total (G), stubby (H), mushroom (I) and thin (J) spine density in DG from control and susceptible mice treatment with ACSF or ARL67156 (n = 11, 13, 10 segments from 2, 4, 2 mice, respectively. For total density: Treatment F2, 31 = 4.474, P = 0.0196; CSDS - ASCF vs Ctrl - ACSF, P = 0.0366; CSDS - ARL67156 vs CSDS - ACSF, P = 0.0086; stubby density: Treatment F2, 31 = 3.327, P=0.0491; CSDS - ASCF vs Ctrl - ACSF, P = 0.1026; CSDS - ARL67156 vs CSDS - ACSF, P = 0.0179; mushroom density: Treatment F2, 31 = 1.986, P = 0.1543; thin density: F2, 31 = 1.475, P = 0.2445. One-way ANOVA with Fisher's LSD test).
Figure 4Figure 4a. Experimental outline. AS CDK12 HCT116 cells were arrested by serum starvation for 72 h and released into the serum-containing medium with (+) or without (-) 3-MB-PP1. 3-MB-PP1 was washed away and replaced with fresh medium at indicated times after the release and samples were subject to RT-qPCR, Western blotting and flow cytometry analyses at 7, 12 and 15 h after the release, respectively. Note that shown wash away time points (2, 3, 4, 5 h) are valid for RT-qPCR only, for Western blotting and flow cytometry 1, 2, 3, 5 h and 1, 3, 5, 7 h wash away time points were applied, respectively. All experiments were performed in at least 3 replicates. Removal of CDK12 inhibitor in early G1/S rescues replication gene expression and cell cycle progression. RT-qPCR of replication gene mRNA RT-qPCR were performed 7, 12 and 15 h post-release, respectively. CTRL=control samples without the 3-MB-PP1. In b, n=3 and error bars indicate SEM. In c, d representative images from 3 biological replicates are shown. Removal of CDK12 inhibitor in early G1/S rescues cell cycle progression. Western blotting of , protein levels Western blotting were performed 7, 12 and 15 h post-release, respectively. CTRL=control samples without the 3-MB-PP1. In b, n=3 and error bars indicate SEM. In c, d representative images from 3 biological replicates are shown. Removal of CDK12 inhibitor in early G1/S rescues replication and cell cycle progression. flow cytometry analyses of cell cycle progression, flow cytometry analyses were performed 7, 12 and 15 h post-release, respectively. CTRL=control samples without the 3-MB-PP1. In b, n=3 and error bars indicate SEM. In c, d representative images from 3 biological replicates are shown. Figure 4e. Rescued loading of CDC6 and CDT1 on chromatin after removal of CDK12 inhibitor. Western blot analyses of chromatin fractions of serum starved AS CDK12 HCT116 cells treated with 3-MB-PP1 for 6 or 9 h or with the inhibitor washed off after 1 h of treatment. CTRL corresponds to cells not treated with the inhibitor at the time of the serum addition. All cells were harvested either 6 or 9 h after the serum addition. Histone H2A serves as a loading control of chromatin fractions and studied DNA replication factors are indicated. A representative image of three replicates is shown. Figure 4f. Inhibition of CDK12 kinase activity in cycling cells leads to decreased numbers of actively replicating cells. Asynchronous AS CDK12 HCT116 cells were grown for 24 and 48 h in the presence or absence of 3-MB-PP1 and replicating BrdU-stained cells were quantified by FACS analyses. CTRL=control samples without the 3-MB-PP1. A representative image of 3 replicates is shown. Figure 4g, h. Prolonged CDK12 inhibition causes chromosomal aberrations in cells. Specific chromosomal aberrations in cells treated with 3-MB-PP1 (24 or 48 h), 4 mM hydroxyurea (5 h) or control solvent (CTRL) were identified by microscopy. A representative image from 3 biological replicates is shown (g). Total numbers of chromosomal aberrations per hundred cells of the representative replicate in g is quantified (h).
Figure 2(A) Internalization and processing of DQ™ Green BSA by uninfected and S. aureus (in blue) infected RAW 264.7 macrophages at 10 h post-infection. These images are representative of three independent experiments. (B) Quantitation of DQ BSA green fluorescence displayed by either uninfected or S. aureus (in blue) infected RAW 264.7. Shown is the mean fluorescence intensity (MFI) in arbitrary units (a.u) ± standard deviation where each symbol represents the measurement of a single cell. The data derive from three independent experiments and n.s. indicates the data are not significant as determined by an unpaired two-tailed t-test (p>0.05). (C) Fluorescence-based proliferation assays were performed in conjunction with the probe DQ™ Green BSA. Processed DQ™ Green BSA could be found co-localizing with intracellular mCherry-expressing S. aureus (in red) and with intracellular S. aureus has replicated (i.e. is proliferation dye negative) at 10h post-infection as depicted here. Data information: Scale bars equal 10 μm.
PolII ChIL dissects the transcriptional activity of genes in breast cancer tissue (A) IGV screen shots of PolII S5P ChIL signals are shown for representative genes in breast cancer tissues.Figure 5(A) IGV screen shots of PolII S5P ChIL signals are shown for representative genes in breast cancer tissues. For each lane, individual samples were selected from each progressive stage of breast cancer. (B) Gene set enrichment analysis of the CGP (chemical and genetic perturbations) gene set in MsigDB. The dots indicate the estimated NES using log2FC to the normal samples in TSS level and TPM. Terms that satisfy BH FDR<0.1 are shown. P-values were estimated by the adaptive multi-level split Monte Carlo scheme implemented in the fgsea R package. (C) Expression, TSS level, and TR of genes. EEF1A1 is shown as a control housekeeping gene. The horizontal dotted line indicates the median level of all genes. Error bars indicate 95% confidence intervals for each estimate indicated by dots (N=1 for each).
ERK7 regulates nucleolar size and ribosome biogenesisA.Figure 5A. Representative immunofluorescent images of control and ERK71 mutant fat bodies with DAPI staining to visualize nucleus. Scale bar: 30 µm. B, C. ERK71 mutant fat bodies display increased nuclear area (B, N>160 cells, obtained from 5 independent fat bodies) and increased nucleolar/nuclear area ratio (C, N>80 cells for nucleolus/nucleus ratio, obtained from 5 independent fat bodies), when compared to control fat bodies. D-F. ERK7 expressing fat body clones (D, marked by GFP, scale bar: 20 µm) display reduced nuclear area (E) and reduced nucleolar/nuclear area ratio (F), when compared to control cells. Nuclei were visualized by DAPI staining, nucleoli were visualized by anti-Fibrillarin antibodies (N=10). G, H. Expression analysis of Pol I (G) and Pol III (H) target RNAs by qRT-PCR. Expression of CDK7 was used for normalization (N=3 replicates of 10 fat bodies/replicate for each genotype).
Figure 3(A) Sequences of the Nrxn1β mutants used, color-coded as in Fig 1A, with residues previously mapped to bind CA10 indicated. Right, table summarizing properties of the recombinant variants: 'HS?' indicates if HS-modified when expressed alone (Fig 1B), 'Binds CA10?', indicates if found to bind CA10, according to (et al,Sterky 2017), and whether the HS modification is blocked by CA10 (this figure). Asterisk denotes non-covalent interaction (see discussion). (B) Representative immunoblot of the indicated proteins harvested from HEK293 cell media and subjected to heparinase treatment ('Heps'). Samples were analyzed by reducing SDS/PAGE and immunoblotting with antibodies against Fc (for Nrxn1β), the 3G10 epitope (for the HS stub) and V5 (for CA10). Input samples were analyzed separately for CA10 to ensure equal expression (lower panel). (C) Quantification of the 3G10 epitope signal, normalized to Fc (Nrxn1β). Data shown are mean ±SEM of 3 independent replicates. *, p< 0.05; by Mann-Whitney tests comparing each Nrxn1 mutant with and without CA10 (n=3). No significance difference was observed for S&T>G (p=0.1143), GIGG (p=0.9714) or DIRR (p=0.4857) variants. Fc-tagged (secreted) Nrxn1β and HA-tagged (transmembrane) Nrxn1α were co-expressed with CA10 and a CA10 mutant for the reactive cysteine CA10(C310A). (D) Summary of CA10 variants used, and their properties (this figure). Asterisk denotes non-covalent interaction (see discussion). (E) representative immunoblots analyzed using antibodies against the Fc-tag (for Nrxn1β-Fc) the 3G10 epitope (for the HS stub) and V5 (CA10-V5), as indicated. CA10 in input samples is shown for demonstration of equal expression. (F, Quantification of the 3G10 epitope signal normalized to Fc (for Nrxn1β-Fc) signals, Data shown are mean ±SEM of 3 biological replicates. *, p< 0.05; ***, p<0.001 by 2-way ANOVA and Holm-Sidak tests. (G) show representative immunoblots analyzed using antibodies against the HA tag (Nrxn1α-HA), the 3G10 epitope (for the HS stub) and V5 (CA10-V5), as indicated. CA10 in input samples is shown for demonstration of equal expression. H) Quantification of the 3G10 epitope signal normalized to HA (for Nrxn1α-HA) signals, respectively. Data shown are mean ±SEM of 3 biological replicates. *, p< 0.05; ***, p<0.001 by 2-way ANOVA and Holm-Sidak tests.
Figure 7(A) ADP-Glo ATPase assay showing stimulation of S. cerevisiae Hrr251-394 (blue circles), Hrr251-394 K38R (red diamonds), Hrr25 full-length:Mam187-191 (black triangles), Hrr251-394:Mam187-191 (orange squares), and Hrr251-394:Mam187-191 R131A (green triangles) by bovine casein. See Figure EV6A for SDS-PAGE analysis of purified proteins. (B) Stimulation of Hrr251-394, Hrr251-394:Mam187-191, and Hrr251-394:Mam187-191 R131A by ATP, measured using an enzyme-coupled ATPase assay. (C) ADP-Glo assay showing the effect of added CK1-7 on Hrr251-394, Hrr251-394:Mam187-191, and Hrr251-394:Mam187-191 R131A. (D) Km, Vmax, and IC50 values for tested enzymes. Vmax was calculated from curves in (A) at 100 M ATP.
Figure 6(A, B) Exercise capacity of MCK-KO and WT mice treated with PBS (vehicle) or NAC (200mg/kg, i.p). (n = 4 WT and n = 6 MCK-KO , mean ±SEM, * p < 0.05, two-tailed student's t-test and two-way ANOVA). (C) Hydrogen peroxide (H2O2) levels (n = 6 for Control groups and Dnmt3a KD with NAC treatment group n = 4 Dnmt3a KD, means ± SEM, * p < 0.05, two-tailed student's t-test and two-way ANOVA followed by Bonferroni post-hoc testing). (D, E) mitochondrial respiration in Dnmt3a knockdown and control L6 myotubes treated with NAC (3mM) or vehicle treatment for 24hrs (n = 5 Control groups n = 3 Dnmt3a KD, n = 4 Dnmt3a KD with NAC treatment groups. means ± SEM, * p < 0.05, two-tailed student's t-test and two-way ANOVA followed by Bonferroni post-hoc testing).
Figure 6A Western blot analysis (probing with an anti-V5 mAb) to detect degradation of N-terminally V5-tagged wild-type (WT) or modified (P300A) HybA in S. sonneiΔrhom7 chromosomally expressing wild-type (+) or inactive (S201A) GlpG with (+) or without (-) HybB. Rhomboid substrates that are uncleaved, cleaved by GlpG, , are marked by black, red, arrows, respectively. Degradation products post rhomboid cleavage are marked by green arrows. B Degradation of V5-tagged HybA at times after blocking protein translation at T0 in the presence (+) or absence (-) of HybB. Rhomboid substrates that are uncleaved, cleaved by GlpG, black, red, arrows, respectively. Degradation products post rhomboid cleavage are marked by green arrows. C Western blot analysis of N-terminally V5-tagged wild-type (WT) or modified (P259A) FdoH in S. sonneiΔrhom7 chromosomally expressing wild-type (+) or inactive (S201A) GlpG with (+)/without (-) FdoI. Rhomboid substrates that are uncleaved, cleaved by GlpG are marked by black, red, arrows, respectively. D Western blot analysis of N-terminally V5-tagged wild-type (WT) or modified (P259A) FdnH in S. sonneiΔrhom7 with wild-type (+) or inactive (SAHA) Rhom7 expressed from pBAD33 with (+)/without (-) FdnI. Rhomboid substrates that are uncleaved, cleaved by Rhom7, are marked by black, blue arrows, respectively. Degradation of N-terminally V5-tagged wild-type (WT) or modified (P259A) FdoH in S. sonneiΔrhom7 with wild-type (+) or inactive (S201A, SAHA) GlpG expressed chromosomally (native) or from pUC19 (plasmid) without FdoI , +/- exposure to 400 µM CuCl2 for 30 min. : Rhomboid substrates that are uncleaved, cleaved by GlpG, are marked by black, red, arrows, respectively. Degradation products post rhomboid cleavage are marked by green arrows. F Degradation of N-terminally V5-tagged wild-type (WT) or modified (P259A) FdnH in S. sonneiΔrhom7 with wild-type (+) or inactive (S201A, SAHA) GlpG expressed from pUC19 (plasmid) without FdnI (-), , +/- exposure to 400 µM CuCl2 for 30 min. Rhomboid substrates that are uncleaved, cleaved by Rhom7, are marked by black, and blue arrows, respectively. Degradation products post rhomboid cleavage are marked by green arrows.
Fig.Figure 4(A, B) ELISA analysis of IL6 in HeLa NOD1 cells (A) or HeLa NOD2 cells (B) upon indicated stimulations for 20 h. For cytosolic delivery of S1P, iE-DAP or MDP, cells were treated together with digitonin (5 µg/ml). (C) GFP immunoprecipitation (IP) of NOD1/2 upon S1P, iE-DAP or MDP stimulation. S1P (20 µM), iE-DAP (20 µM) or MDP (20 µM) were delivered into the cytosol of HeLa NOD1 or HeLa NOD2 cells with digitonin (5 µg/ml) for 1 h. Afterwards cell lysates were collected for IP. (D) Western blot analysis of MAPKs and NF-κB activation upon S1P stimulation. HeLa NOD1 or NOD2 cells were treated with cytosolic S1P (20 µM), iE-DAP (20 µM) or MDP (20 µM) for indicated times. (E) NF-κB luciferase assay in HEK293T WT and NOD1/2 KO NF-κB reporter cells upon cytosolic S1P stimulation. (F) qRT-PCR analysis of IL8 expression upon cytosolic S1P stimulation in HEK293T WT and NOD1/2 KO cells. (G) qRT-PCR analysis of Il6 expression in WT, Nod1/2 and Rip2 KO BMDMs upon S1P (10 µM) or MDP (10 µM) stimulations together with digitonin (2.5 µg/ml) for 4 h. Multiplex analysis of IL6 (H) production in supernatants of WT, Nod1/2 and Rip2 KO BMDMs upon S1P (10 µM) or MDP (10 µM) stimulations together with digitonin (2.5 µg/ml) for 20 h. Multiplex analysis of CXCL2 (I), CCL5 (J) and IL16 (K) production in supernatants of WT, Nod1/2 and Rip2 KO BMDMs upon S1P (10 µM) or MDP (10 µM) stimulations together with digitonin (2.5 µg/ml) for 20 h.
Lysosomal stress induces TFEB nuclear translocation.Figure 1(A) Immunofluorescence of HEK‐293T cells that express TFEB-3 × FLAG, subjected to the indicated treatments and stained with antibodies against FLAG and the lysosomal marker LAMP2. The FLAG and LAMP2 channels are in green and red, respectively, in the merge. DAPI (blue) is included in the merge. Scale bars represent 10 μm. (B) Quantification of the number of cells with nuclear TFEB-3 × FLAG in the four conditions in (A). Each value represents mean±s.d. from three independent fields with N=300. (C) Immunofluorescence of HEK‐293T cells treated as indicated and stained with antibodies against endogenous TFEB and the lysosomal protein RagC (green and red, respectively, in the merge). DAPI is included in the merge. Scale bars represent 10 μm. (D) Immunoblotting of proteins extracted from HeLa cells that express TFEB-3 × FLAG treated with DMSO, chloroquine (CQ) or SalA, subjected to nuclear/cytosolic fractionation and blotted with antibody against FLAG to detect TFEB. H3 and tubulin were used as nuclear and cytosolic markers, respectively. Blots are representative of triplicate experiments.
miR-155 decreases mycobacterial survival in macrophages.Figure 2(A-C) RAW264.7 cells were transfected with control or miR-155 mimic for 24 h followed by BCG (A), H37Ra (B) or H37Rv (C) infection at an MOI of 10 for 1 h, and intracellular mycobacterial viability was determined by CFU assay at the indicated timepoints. (D-F) RAW264.7 cells were transfected with control or miR-155 inhibitor for 24 h followed by BCG (D), H37Ra (E) or H37Rv (F) infection at an MOI of 10 for 1 h, and intracellular mycobacterial viability was determined by CFU assay at the indicated timepoints. (G and H) RAW264.7 cells were transfected with miR-155 mimic (G) or inhibitor (H) for 24 h followed by Texas Red-labeled BCG infection at an MOI of 10 for 1 h. Phagocytosis of mycobacteria was determined by flow cytometry. Data are shown as the mean ± SEM of three independent experiments. *, p<0.05; **, p<0.01.
Figure 3Female C57BL/6J mice were put in normoxia or hypoxia for 6h or 24h, injected i.p. with PBS or DEX (10 mg/kg) and livers were isolated for genome-wide transcriptomics via RNA-seq. N=3 per group for a single RNA-seq. (A, B) Examples of DEX-responsive genes in hypoxia 6h (A) and 24h (B) which were not induced by DEX in normoxia. Data information: All bars represent mean ± SEM. P-values were calculated using two-way ANOVA followed by post-hoc Šídák's multiple comparisons test to correct for multiple testing during the pairwise multiple comparisons, except if otherwise stated. ***P<0.001, **P<0.01, *P≤0.05. ChIP-seq on liver derived from mice which were subjected to hypoxia (6h and 24h), followed by DEX (10 mg/kg) injection and 2h later sacrificed for liver isolation. (C) Box plots showing the LFC of genes responsive to DEX in hypoxia, but not in normoxia (6h and 24h). The central band represents the median. The box ranges from Q1 to Q3 which represents the interquartile range (IQR = Q3 - Q1) and covers the central 50% of the data. The whiskers illustrate the minimum (Q1 - 1.5*IQR) and maximum (Q3 + 1.5*IQR) of the data. Outliers are shown as dots. N=3 biological replicates per condition. ChIP-seq on liver derived from mice which were subjected to hypoxia (6h and 24h), followed by DEX (10 mg/kg) injection and 2h later sacrificed for liver isolation. (D) Histogram showing the coverage per position in a region 350 bp up- and downstream of the GR DNA-binding peaks found in hypoxia after PBS or DEX stimulation. ChIP-seq on liver derived from mice which were subjected to hypoxia (6h and 24h), followed by DEX (10 mg/kg) injection and 2h later sacrificed for liver isolation. (E) Examples of specific GR DNA-binding peaks, associated with the DEX-induced gene Slc22a5 in hypoxia (6h and 24h). (F) HIF1aHIF2afl/fl and HIF1aHIF2aAlbKO mice were put in hypoxia for 6h and stimulated with DEX. Slc25a30 expression was measured in the liver via RT-qPCR. N=3 per group, one experiment. Data information: All bars represent mean ± SEM. P-values were calculated using two-way ANOVA followed by post-hoc Šídák's multiple comparisons test to correct for multiple testing during the pairwise multiple comparisons, except if otherwise stated. ***P<0.001, **P<0.01, *P≤0.05.