EMBO/sd-character-level-ner huggingface dataset:
EXO1, but not BLM, WRN or DNA2, promotes LIG3‐dependent end‐joining of telomeres uncapped by TRF2 inactivation A–C MEFs of the indicated genotypes were infected with retroviruses expressing the indicated shRNAs, followed by selection with puromycin for 72 h.Figure 6A-C MEFs of the indicated genotypes were infected with retroviruses expressing the indicated shRNAs, followed by selection with puromycin for 72 h. Mitotic chromosomes isolated 48 h later were fixed and stained with a Cy3‐conjugated (CCCTAA)3‐PNA probe. The frequency of end‐to‐end chromosome‐type fusions is represented as a percentage of fusions observed after TRF2 depletion in each experiment. A minimum of 1,200 chromosomes were scored for each sample. Error bars represent SD of two independent experiments. P‐values were calculated using an unpaired two‐tailed t‐test. *P ≤ 0.05; NS, P > 0.05. Each graph represents a separate set of experiments. A-C MEFs of the indicated genotypes were infected with retroviruses expressing the indicated shRNAs, followed by selection with puromycin for 72 h. Mitotic chromosomes isolated 48 h later were fixed and stained with a Cy3‐conjugated (CCCTAA)3‐PNA probe. The frequency of end‐to‐end chromosome‐type fusions is represented as a percentage of fusions observed after TRF2 depletion in each experiment. A minimum of 1,200 chromosomes were scored for each sample. Error bars represent SD of two independent experiments. P‐values were calculated using an unpaired two‐tailed t‐test. *P ≤ 0.05; NS, P > 0.05. Each graph represents a separate set of experiments. A-C MEFs of the indicated genotypes were infected with retroviruses expressing the indicated shRNAs, followed by selection with puromycin for 72 h. Mitotic chromosomes isolated 48 h later were fixed and stained with a Cy3‐conjugated (CCCTAA)3‐PNA probe. The frequency of end‐to‐end chromosome‐type fusions is represented as a percentage of fusions observed after TRF2 depletion in each experiment. A minimum of 1,200 chromosomes were scored for each sample. Error bars represent SD of two independent experiments. P‐values were calculated using an unpaired two‐tailed t‐test. *P ≤ 0.05; NS, P > 0.05. Each graph represents a separate set of experiments.
Figure 6a mCherry-tagged truncated BRD4 mutant proteins and GFP-tagged ISX were detected in anti-GFP immunoprecipitates by Western blot. Data information: Each experiment was repeated at least three times. The DNA binding activity of Snail (b) were evaluated in anti-GFP-mCherry ChIP-ChIP immunoprecipitates by RT-PCR in A549 cells. Red, Hprt1 promoter (-190-+40 bp, negative control). (10% input of each groups were pull down and applied to qPCR) Data are presented as mean ± SD in bar graph (p < 0.001, Student's t‐test) of 3 independent experiments, each performed in triplicate. Data information: Each experiment was repeated at least three times. c, The DNA binding activity of TWIST1 (c) were evaluated in anti-GFP-mCherry ChIP-ChIP immunoprecipitates by RT-PCR in A549 cells. Red, Hprt1 promoter (-190-+40 bp, negative control). (10% input of each groups were pull down and applied to qPCR) Data are presented as mean ± SD in bar graph (p < 0.001, Student's t‐test) of 3 independent experiments, each performed in triplicate. Data information: Each experiment was repeated at least three times. The cell migration (wound healing, d) activity were determined in A549 cells co-transfected with cDNAs for GFP-tagged ISX and mCherry-tagged BRD4 mutants. Data are presented as mean ± SD in bar graph (p < 0.001, Student's t‐test) of 3 independent experiments, each performed in triplicate. Data information: Each experiment was repeated at least three times. e, The cell invasion (transwell, e) activity were determined in A549 cells co-transfected with cDNAs for GFP-tagged ISX and mCherry-tagged BRD4 mutants. Data are presented as mean ± SD in bar graph (p < 0.001, Student's t‐test) of 3 independent experiments, each performed in triplicate. Data information: Each experiment was repeated at least three times.
Figure 3Frequencies of FITC+ THP-1 cells stimulated with FITC-ecGAMP for 1 h in presence of DMSO or indicated inhibitors including Dynasore (10 μM), chlorpromazine (CPZ, 10 μM), dimethylamiloride (DMA, 100 μM), polyinosinic acid (Poly I, 50 μg/ml) or mannans from Sacharomyces cerevesiae (Mannans, 1 mg/ml). qRT-PCR detection of IFNB1 (B) and IL6 (C) mRNA in THP-1 cells stimulated with ecGAMP (5 μg/ml) or intracellular 2′3′-cGAMP (icGAMP) (0.1 μg/ml) for 4 h in presence of DMSO or dynasore (10 μM). qRT-PCR detection of Ifnb1 (D) and Il6 (E) mRNA in mBMDMs stimulated with ecGAMP (5 μg/ml) or icGAMP (0.1 μg/ml) for 4 h in presence of DMSO or dynasore (10 μM). Immunostaining for EEA1 (red) (F) and LAMP2 (red) (G) in THP-1 cells stimulated with FITC-ecGAMP (5 μg/ml, green) for 30 min, nucleus in blue (DAPI). Data are representative of 3 independent experiments. Scale bar, 10 μm. qRT-PCR detection of IFNB1 and IL6 mRNA in THP-1 cells (H, I) and mBMDMs (J, K) stimulated with ecGAMP (5 μg/ml) and icGAMP (0.1 μg/ml), respectively, for 4 h in presence of DMSO or bafilomycin A1 (BafA) (1 μM).
Figure 3(A) Whole-cell lysates from control and patient (case index, CI) fibroblasts were assessed by 3-12% native-PAGE gradient gels with proteasome bands (30S, 26S and 20S complexes) visualized by their ability to cleave the Suc-LLVY-AMC fluorogenic peptide. (B) Ten micrograms of control and patient cell lysates were tested for their chymotrypsin-like activity by incubating them with 0.1 mM of the Suc-LLVY-AMC substrate at 37°C over a 180-h period of time in quadruplicates on a 96-well plate. Indicated on the y-axis are the raw fluorescence values measured by a microplate reader and reflecting the AMC cleavage from the peptide. Bars show the mean of 4 independent experiments +/- SD. (C) Proteasome complexes from control and patient fibroblasts separated by native-PAGE were subjected to western-blotting using antibodies specific for α6, Rpt5 (PSMC3) and PA28-α, as indicated. (D) Proteins extracted from control and CI PSMC3 were separated by 10 or 12.5% SDS-PAGE prior to western-blotting using primary antibodies directed against ubiquitin and several proteasome subunits and/or components including α6, β1, β; β5, β5i, Rpt2 (PSMC1), Rpt5 (PSMC3), Rpt3 (PSMC4), Rpt4 (PSMC6) and PA28-α, as indicated. For the PSMC3 staining, two exposure times are shown. Arrow indicates an additional PSMC3 species corresponding to the expected size of the truncated PSMC3 variant. Equal protein loading between samples was ensured by probing the membrane with an anti-α-Tubulin antibody.
Figure 5(A) Immunocytochemistry showing post-synaptic marker PSD-95 (red), pre-synaptic marker Synapsin-I (green), dendritic marker Map2 (blue) and co-localization of PSD-95 and Synapsin-I from Nedd4-2 WT or Nedd4-2 cKO cultured hippocampal neurons at DIV 16 treated with TAT, TAT-S3, or TAT-pS3 peptide for 12 hrs (10 μM). Representative secondary dendrites of Nedd4-2 WT (top left) and Nedd4-2 cKO (bottom left) neurons are shown. Quantification of colocalized synaptic puncta number and synaptic puncta area for Nedd4-2 WT (top right) and Nedd4-2 cKO (bottom right) neurons is on the right (n = 32-36 neurons). Scale bar: 5 µm. (B) Patch-clamp recordings from Nedd4-2 WT or Nedd4-2 cKO hippocampal neurons treated with TAT, TAT-S3, or TAT-pS3 peptide for 12 hrs (10 μM) at DIV 14-17. Representative mEPSC traces from Nedd4-2 WT (top left) and Nedd4-2 cKO (bottom left) are shown. Quantification of mEPSC amplitude and frequency from Nedd4-2 WT (top right) and Nedd4-2 cKO (bottom right) neurons are shown (n = 22-24 neurons). (C) fEPSP recording from Nedd4-2 WT (left; n = 7 slices from 6 mice for TAT and n = 6 slices from 6 mice for TAT-pS3) and Nedd4-2 cKO (right; n = 9 slices from 9 mice for both TAT and TAT-pS3) hippocampal slices of mice at age 6-8 weeks. The slices were pretreated with TAT or TAT-pS3 peptide (20 μM) for 1 hr before the induction of L-LTP at Schaffer collateral synapses. Representative fEPSP traces were recorded before and 180 min after LTP induction (Scale bars: 0.5 mV, 5 ms). Summary bar graphs showed the fEPSP slopes measured 160-180 min after L-LTP induction at Schaffer collateral synapses.
Figure 5(A) BRET experiment in parental HEK293-AT1 cells in OSW1-pretreated cells after RNAi mediated knock-down of PIP5Kβ or PIP5Kγ. (means ± S.E.M. from three experiments, each performed in triplicates). Note the reduced response in the PIP5Kγ knock-down cells and the slight reduction in the case of PIP5Kβ knock-down. (B) Areas below the curves calculated from the time of rapamycin addition for each of the three separate experiments shown in panel A (means ± S.E.M., n=3). One-way ANOVA with Dunnett's multiple comparisons was used for statistical analysis (* P=0.0241; **P=0.0088). ( (C) These reductions are proportional to the reduction in PIP5Kγ levels as assessed by Western blot analysis of cell lysates obtained from cells treated with siRNAs at the same time. (D) Increased appearance of GFP-LC3 positive vesicles in PI4K2A K/O cells and reduced acidification of the LC3-positive vesicles using the GFP-mCherry-LC3 reporter. Cells were transfected with the indicated constructs for one day and observed by confocal microscopy. Scale bars: 20 µm. (E) Quantification of the LC3 positive vesicles. (n = 67, 34, 26, 13, 30, 27 for the various groups, respectively from left to right). *** designates P < 0.0001 using unpaired t-test. The same data set was also analyzed with the non-parametric Mann-Whitney test showing a P = 0.0004 and 0.0012 for #20 and #26 clones relative to control, respectively. (F) Co-localization analysis using the Pearson coefficient (n = 31, 21 and 14 cells analyzed for the different groups from left to right). *** designates P < 0.0001 using unpaired t-test. The same data set was also analyzed with the non-parametric Mann-Whitney test showing a P value < 0.0001 for both groups.
a) HACE1.f1(a) HACE1 mRNA expression is markedly increased in ischaemic and dilated cardiomyopathy (ICM and DCM) heart failure (HF) patient heart tissue compared with non-failing hearts (NF). Expression levels were normalized to HPRT and GAPDH mRNAs. n=15 per group. Control, non-failing heart. (b) Elevated Hace1 mRNA expression in sTAC mousehearts compared with sham controls, expression levels were normalized to Hprt and Gapdh mRNAs. n=6-9 per group, P=0.004 (unpaired two-tailed Student's t-tests). (c-e) Hace1−/− and WT mice were subjected to sTAC or sham surgery and analysed 4 days after the operation. Echocardiography measurement of (c) LV fractional shortening (FS=(LVEDD−LVESD)/LVEDD × 100%); (d) LV end-diastolic dimension (LVEDD) (mm) and (e) LV end-systolic dimension (LVESD) (mm) are shown. n=6-8 for each group. (f) Mortality rate of WT and Hace1−/− mice after sTAC is shown by Kaplan-Meier plots as the survival proportion of each cohort of mice. Survival incidence in mice over the indicated follow-up period is shown as the ratio of the number of mice survived/total mice analysed for each group. This represents 60% (12/20) for Hace1−/− sTAC mice, 84% (16/19) for Hace1+/+ sTAC mice and 100% (12/12) for both Hace1−/− and Hace1+/+ sham mice on day 4; P value as indicated (log-rank χ2-test). (g) Heart weight/body weight ratios (HW/BW) and (h) lung weight/body weight ratio (LW/BW) of WT and Hace1−/− mice 4 days after sTAC or sham surgery. n=15-21 for each group. (i) Representative haematoxylin and eosin staining transverse heart cross-sections showing myocyte cross-sectional area (scale bars, 20 μm). (j) Real-time RT-PCR analyses reveal a significant increase in the expression of the atrial natriuretic peptide gene (Nppa), a clinical biomarker for heart hypertrophy, in Hace1−/− sTAC heart; expression levels were normalized to Hprt and Gapdh mRNAs. n=6-9 per group. In all panels, error bars represent s.e.m., one-way analysis of variance were used to calculate P-values.
Figure 3Western blots showing ASC-oligomerisation after DSS-mediated crosslinking in insoluble fractions from THP1 cell lysates. PMA differentiated THP1 cells were LPS-primed (1 μg/mL, 4 hrs.) and pre-incubated for 15 min with either 0.1% DMSO or P22077 (2.5 µM). followed by treatment with nigericin (10 μM, 45 mins.). The 'input' represents the abundance of ASC in cell lysates prior to isolation of the insoluble fraction. Blots show membranes that were probed with an anti-ASC antibody. Data is representative of at least three independent experiments. Western blots showing ASC-oligomerisation after DSS-mediated crosslinking in supernatants from THP1s treated as in A. Blots show membranes that were probed with an anti-ASC antibody. The input represents non-crosslinked supernatant. Data is representative of at least three independent experiments. Images from an immunofluorescence experiment showing ASC-speck formation in THP1 cells treated as in A. All images show cells that were stained with an anti-ASC antibody. Images are representative of 3 independent experiments. Scale bar 20µm. Quantification of ASC-speck formation in images depicted in C. The number of ASC specks was quantified and expressed as the percentage of cells containing specks.
Figure 2(A) Anti-acetyllysine Western blot analyses and total protein stains of E. coli cell extracts. Cellular protein extracts before and after induction of His6-MBP-GNAT expression were separated on 12 % acrylamide gels and immunoblotted by using an anti-acetyllysine antibody or stained with Coomassie Dye. GNAT1, 2, 5, 6, 7 and 10 as well as His6-MBP control were expressed in E. coli BL21(DE3) pLysS. GNAT3 and 4 were expressed in Rosetta(DE3). In the total protein stains, recombinant GNAT protein constructs were highlighted by blue arrowheads. Protein expression before (ni) and after (i) induction with IPTG is indicated. The luminescence signal, indicating acetylated proteins, was usually recorded after 40-120 seconds. Since MBP-GNAT4 expression resulted in a saturated signal, the luminescence was additionally recorded for 10 seconds (indicated by an asterisk). (B) Sequence logos of all unique lysine acetylation sites after GNAT expression in E.coli. Uniprot E.coli protein sequences were used as background population (sequence logos were generated using iceLogo
Figure 4(A) Schematic diagram of thalidomide metabolites by CYPs. (B) In vitro binding assay for thalidomide and 5-hydroxythalidomide. Interaction between bls-CRBN and FLAG-GST-IKZF1, -SALL4 and -PLZF in the presence of DMSO, thalidomide or 5-hydroxythalidomide (3.125, 6.25, 12.5, 25, 50, 100 or 200 µM) was analysed using the AlphaScreen technology. All relative AlphaScreen (AS) signals are expressed as a relative luminescent signal with a luminescent signal of DMSO as one. Error bars denote ± standard deviation (n = 3). (C) Immunoblot analysis of AGIA-IKZF1, AGIA-SALL4 or AGIA-PLZF in FLAG-CRBN expressing CRBN-/- HEK293T cells treated with DMSO, thalidomide or 5-hydroxythalidomide for 16 h. (D) Immunoblot analysis of endogenous SALL4 or PLZF protein levels in HuH7 cells treated with DMSO, thalidomide or 5-hydroxythalidomide for 24 h. (E) Immunoblot analysis of endogenous PLZF or IKZF1 protein levels in THP-1 cells treated with DMSO, thalidomide or 5-hydroxythalidomide for 24 h. Data information: Data in bar graphs in (D) and (E) were calculated as relative PLZF band intensity with PLZF band intensity of DMSO as 100. Error bars donated ± standard deviation (n = 3) and P-values were calculated by one-way ANOVA with Tukey's post-hoc test (NS = Not Significant, *P < 0.05, **P < 0.01 and ***P < 0.001). (F) Immunoblot analysis of endogenous SALL4, PLZF, ZFP91 or CK1α protein levels in HuH7 cells treated with DMSO, thalidomide (Tha), lenalidomide (Len), pomalidomide (Pom) or 5-hydroxythalidomide (5-HT) for 24 h.
Celf2a inactivation through CRISPR/Cas9 in UP∆44 myocytes induces skipping of exon 45.Figure 1A GS∆44 myoblasts were transfected with an empty vector (CTRL) or with a CELF2a overexpressing construct (OE CELF2A) (Martone et al, 2016). Cells were collected after 10 days in differentiation medium (DM) and the RNA was analysed by RT-PCR for CELF2a expression and DMD exon 45 skipping. GAPDH was used as loading control. The arrow indicates the DMD exon 45 skipped band. Representative results are shown (n=3). B Representative immunofluorescence for Myosin Heavy Chain (MHC in red) in combination with DAPI staining (in blue) of iPSCs obtained from WT (WT#1), UP∆44 (UP∆44#3) and ∆44 edited clones (UP∆44#3.1 and UP∆44#3.5) differentiated for 9 days into myocytes by MYOD/BAF60c overexpression. Scale bar 100 μm. C Histogram represents fusion index quantification. At least 7 randomly chosen microscope fields of two independent biological samples were analysed (n=2). Data are presented as mean ± SD of the biological replicates. D RNA extracted from myocytes obtained by differentiation of WT (WT#1), UP∆44 (UP∆44#3) and edited UP∆44 iPSC (UP∆44#3.1 ∆CELF2a and UP∆44#3.5 ∆CELF2a) clones was analysed by RT-PCR for CELF2a, CELF2b, CELF2c and DMD exon 45 skipping. GAPDH was used as loading control. The arrow indicates the DMD exon 45 skipped band. Representative results are shown (n=3). E Western blot on proteins (40 μg) extracted from myocytes obtained by differentiation of WT (WT#1), UP∆44 (UP∆44#3) and edited UP∆44 iPSC (UP∆44#3.1 ∆CELF2a and UP∆44#3.5 ∆CELF2a) clones probed with antibodies against dystrophin (DMD). Actinin (ACTN) was used as a loading control. For the WT 5% sample 2 μg of proteins were diluted in 38 μg of proteins belonging to UP∆44#3 to reach a total amount of 40 μg. Representative results are shown (n=3).
Figure 2(A) Clusters of gallbladder tissue (GT)-specific, liver tissue (LT)-specific and small intestine tissue (IT)-specific genes (Table EV2). The number of genes and the names of selected genes in each cluster are indicated. Gene expression levels (log2 scale) are shown for gallbladder organoids (GO), liver organoids (LO), intestinal organoids (IO) and gallbladder (GT), liver (LT) and intestinal (IT) tissues. (B) Genes expressed in gallbladder and liver tissues, but not in the intestine, divided into two subclusters, depending on whether the genes were expressed or not in gallbladder organoids (Table EV3). The number of genes and the names of selected genes in each subcluster are indicated. (C) Clusters of organoid (O)-specific and tissue (T)-specific genes (Table EV4). The number of genes and the names of selected genes in each cluster are indicated. (D) Expression levels of selected genes from the clusters shown in panels B and C. G, gallbladder; L, liver; I, intestine; O, organoid; T, tissue.
Figure 5A, C Representative live-cell STED super-resolution images (t=0 s) showing WT (A) and MIC13 KO (C) Hela cells expressing MIC10-SNAP stained with silicone-rhodamine. Box in (A and C) mark selection shown as a zoom in panel (B and D) respectively. Scale bar 500 nm.E Blind quantification of merging and splitting events of CJs in WT and MIC13 KO Hela cells expressing MIC10-SNAP (3 independent experiments, 5-8 mitochondria for each experiment) represented as boxplots. Boxplots show median and interquartile range from 25 to 75 percentile and whiskers represent minimum and maximum value. *P = 0.026 (merging and splitting events in WT cells) and ****P < 0.0001 (merging and splitting events of WT vs. MIC13 KO), unpaired Student's t-test. F Scheme representing the dynamic nature of CJs in WT and MIC13 KO cells.
Figure 1A. The core histones and overview of the corresponding QPrEST and peptide standards mapped out on the protein sequence. B. Relative quantification of all four histone proteins in each tissue replicate (order of appearance per replicate: H2A, H2B, H3.3 and H4). C. Immunohistochemistry images from the Human Protein Atlas (www.proteinatlas.org) for protein ANXA1 with nuclear staining (blue) for three selected tissues. D. Calibration curves for two of the four histone peptides, with decreasing amount of QPrEST standard spiked into a U2OS cell lysate.
Figure 2(A) Immunoblot analysis. Primary hepatocytes prepared from wild-type mice were infected with adenovirus expressing GFP-Nrf2 and GFP or NBR1 for 48 hrs. Total cell lysates were prepared and subjected to immunoblot analysis with the indicated antibodies. Data shown are representative of three separate experiments. Bar graphs indicate the quantitative densitometric analysis of the indicated proteins relative to actin. Data are shown as means ± s.e. **P < 0.01 as determined by Welch's t-test. (B) Immunoblot analysis. Primary hepatocytes prepared from p62f/f and p62f/f; Alb-Cre mice were infected with adenovirus against GFP or NBR1 for 48 hrs. Cytosolic and nuclear fractions were prepared and subjected to immunoblot analysis with the indicated antibodies. Data shown are representative of three separate experiments. Bar graphs indicate the quantitative densitometric analysis of the indicated proteins relative to actin (cytosolic proteins) or Lamin B (nuclear proteins). Data are shown as means ± s.e. *P < 0.05 as determined by Welch's t-test. (C) Immunofluorescence microscopy. Primary hepatocytes from wild type mice were infected with adenovirus expressing GFP-Nrf2 and LacZ or NBR1 for 48 hrs. More than 100 cells were assayed for the nuclear signal of Nrf2. Data are shown as means ± s.e. ***P < 0.001 as determined by Welch's t-test. Bars: 10 µm. (D) Total RNAs were prepared from primary hepatocytes described in (B). Values were normalized against the amount of mRNA in the p62f/f hepatocytes expressing GFP. The experiments were performed at least three times. Data are are shown as means ± s.e. *P < 0.05 as determined by Welch's t-test.
Clinical response of patient M032 to vemurafenib.Figure 1Treatment schedule of patient M032, who received a PET scan two weeks before the start of the treatment (purple asterisk), followed by a baseline CT scan and every two months a follow-up CT scan (red asterisks). Metastases were surgically removed either shortly before the start of the treatment or after resistance occurred (post, M032R1-R5). PR, partial response; PD, progressive disease. Locations of the removed metastases are indicated in the illustration. Examples of CT scan images of several metastases (M032R1, M032R2, and M032R5). Volumetric measurements based on the PET and CT scans could be generated from M032, M032R1, and M032R5. Graph illustrates that the metastases R1 and R5 initially expanded before the start of the treatment, but regressed upon vemurafenib treatment. M032R5 showed progressive disease after 4 months, whereas M032R1 still displayed stable disease. M032 was excised and no recurrence was observed.
Figure 5Activity phosphorylation of p38 were detected in DCs in the spleens from DC(WT) and DC(NLRC3-KO) mice 26 d after EAE induction. Pooled data are presented in the right panel. DC(p38-KO) and DC(p38+NLRC3-KO) mice were immunized with MOG(35-55) peptide in CFA adjuvant and pertussis toxin to induce EAE. Mean clinical scores of EAE in immunized DC(WT) (n= 5) and DC(NLRC3-KO) mice (n= 5). DC(p38-KO) and DC(p38+NLRC3-KO) mice were immunized with MOG(35-55) peptide in CFA adjuvant and pertussis toxin to induce EAE. Frequencies of CD4+ T cells that express IFN-γ and IL-17A in the spinal cord and brain from DC(WT) and DC(NLRC3-KO) mice 26 d after EAE induction after restimulation with MOG(35-55) peptide. Pooled data are presented in the right panel. DC(p38-KO) and DC(p38+NLRC3-KO) mice were immunized with MOG(35-55) peptide in CFA adjuvant and pertussis toxin to induce EAE. Expression of Il12, Il6 and Il23 mRNA in DCs sorted from spleens of DC(WT), DC(NLRC3-KO), DC(p38-KO) and DC(p38+NLRC3-KO) mice 26 d after EAE induction, presented relative to that of Gapdh.