EMBO/sd-character-level-ner huggingface dataset:
Model KAP1 regulates ERVs and ZNFs in differentiated human cells including adult PBMCs and contributes to innate immune control.Figure 6KAP1 regulates ERVs and ZNFs in differentiated human cells including adult PBMCs and contributes to innate immune control. KAP1 and SETDB1 binding [30] are detected at ERVs and ZNF genes in differentiated cells and overlap with the silent chromatin mark H3K9me3 as well as some cytosine methylation, which we detect at HERVK14C and SVAs. KAP1 inactivation reveals that the KAP1-KZNF pathway is functionally intact and required in differentiated cells including PBMCs. KAP1-depletion leads to a decrease in H3K9me3 at retrotransposons and to reactivation of HERVs and SVAs. These retrotransposons or potentially other endogenous RNAs produce double stranded RNA structures, which activate a type I interferon response through MAVS signaling. KAP1 depletion is not sufficient for global ISG induction, suggesting it exerts partial redundancy at repressing ERVs with other epigenetic mechanisms in differentiated human cells.
Figure 4A Relationship among MXL-1, TDPT-1, ETS-4, CEBP-1 and PKA in the regulation of axon injury-induced svh-2 expression. B Interaction between ETS-4 and TDPT-1. COS-7 cells were transfected with plasmids encoding T7-TDPT-1, HA-ETS-4 (WT), HA-ETS-4(S73E) (SE) and HA-ETS-4(S73A) (SA), as indicated. Whole-cell extracts (WCE) and immunoprecipitated complexes obtained with anti-T7 antibody (IP: T7) were analyzed by immunoblotting (IB). C In vitro SUMOylation of ETS-4. COS-7 cells were transfected with plasmids encoding HA-ETS-4 (WT) and HA-ETS-4(K32A; K83A) (2KA), as indicated. Cell lysates were immunoprecipitated with anti-HA antibody. The immunoprecipitates were subjected to in vitro SUMOylation assays. Reaction mixtures were analyzed by immunoblotting (IB) with anti-SUMO-2/3 and anti-HA antibodies. Arrows mark the SUMOylated-ETS-4 bands. D Schematic representation of the two putative consensus SUMOylation sites and domain structure in ETS-4. Domains are shown as follows: a pointed domain (PNT; yellow) and an Ets DNA-binding domain (ETS; blue). E SUMOylation of ETS-4 in mammalian cells. HEK293 cells were transfected with plasmids encoding HA-ETS-4 (WT), HA-ETS-4(K32A; K83A) (2KA), T7-TDPT-1, His-SUMO-1, His-SUMO-2 and Myc-MXL-1, as indicated. SUMO-conjugated proteins were isolated by cobalt affinity chromatography. ETS-4 was detected by immunoblotting with anti-HA antibody. Whole-cell extracts (WCE) were analyzed by immunoblotting (IB).
Figure 3(A) Glycine region from the 1H-15N HSQC spectrum of [15N,13C] Ubiquitin-maltoheptaose (blue) overlayed with the same region for [15N,13C] Ubiquitin (red). Visible amide assignments are labelled. Arrows indicate the two major chemical shift changes for Gly75 and Gly76 due to conjugation of maltoheptaose to the C-terminus. (B) 1D 1H NMR overlay for the anomeric region of maltoheptaose (red) and [15N,13C] Ub-maltoheptaose (blue) with the 1H-13C HSQC of the latter shown above. Key anomeric assignments are labelled, and arrows indicate the chemical shift perturbations upon conjugation. (C) 1H-13C HMBC NMR spectrum (left) showing a 3-bond connectivity from the C-terminal Gly76 carbonyl 13C to the 6-CH2 proton chemical shifts. Extracted 2D 15N plane (107.9 ppm) from the 3D HN(CA) CO spectrum showing the 1HN amide to 13C carbonyl correlation for Gly76 of ubiquitin. (D) Overlay of the 1H-13C HSQC spectra for free ubiquitin (red) and ubiquitylated-maltose (blue). Assignments for ubiquitin CHα proton are labelled together with the assignments for the ligated and free 6-CH2 positions of the disaccharide labelled. Asterisk indicates a buffer component. (E) Chemical structure of cyclomaltoheptaose (β-cyclodextrin). (F) HOIL-1 activity against the indicated maltosaccharides (2 mM) was assayed and visualised by staining with Coomassie protein stain. Where indicated, assays were treated with 1.5 M hydroxylamine. Met1-linked ubiquitin tetramer was also included in these assays. (G) Chemical structure of 6‑mercapto-cyclomaltoheptaose (heptakis-(6‑deoxy-6‑mercapto)-β‑cyclodextrin). (H) In vitro ubiquitylation of the indicated cyclodextrins (2 mM) by HOIL-1, visualised by Coomassie staining. Met1-linked ubiquitin tetramer was also included in these assays.
VEGF-C is required for lymphatic vessel growth in the developing intestine and for the intestinal lymphatic vessel maintenance in adult mice.Figure 1A-D Mesenteric blood (PECAM1, red) and lymphatic vessels (LYVE1, green; PROX1, gray). Asterisks indicate lymphatic valves, arrow indicates a lymphatic vessel stub, and arrowhead indicates an isolated lymphatic vessel fragmentE-H Blood vessels (PECAM1, green) and lymphatic vessels (VEGFR-3, red) in the small intestinal wall. I, J Detection of chylous ascites (arrows) in the VCiΔR26 mice at P6. K, L LYVE1 staining of intestinal lymphatic vessels at P6. M-T LYVE1 staining of lymphatic vessels in the intestinal wall in adult mice. Genotypes and deletion lengths are indicated in (U).U Quantification of LYVE1 areas in (M-T). Length of the i∆Vegfc gene deletion is indicated in months (mo), and Vegfd indicates the VEGF-D genotype. Data are represented as mean ± SEM. Significant differences were determined using one-way ANOVA and Bonferroni post hoc analysis compared to WT intestine represented in (M). *P = 0.003, **P = 0.001, ***P = 0.0008, ♯P = 0.0002, §P = 0.0001.
Figure 2A Periodic measurement of the oxygen consumption rate (OCR) of mitochondria, isolated from 5 mice per genotype, before and after the administration of the indicated compounds (left). Quantification of OCR in independent experiments, n= 8 per genotype (right). B OCR rates of cardiac mitochondria after administration of pyruvate, malate and ADP, n= 7 per genotype.
Figure 3Figure 3. Decrease of nucleoid number without mtDNA depletion in patient fibroblasts. A. In control and patient fibroblasts, nucleoids were labeled with an antibody against DNA and mitochondria were stained with Mitotracker. Image analysis was performed by confocal microscopy. Scale bar: 10 µm. B-C. The average number (B) and size (C) of nucleoids, labeled with antibodies against DNA shown in A, were quantified for 35 randomly-selected individual cells per each studied fibroblast cell line from 2 independent experiments. Differences between the cell lines were analyzed by Student's t-test (two-sided): extremely significant (***: p<0.001). Nucleoid number (B): patient 1 versus control ***: p=0.0001, patient 2 versus control ***: p=0.001. Nucleoid size (C): patient 1 versus control ***: p=0.0001. D. Mitochondrial DNA content in control and patientfibroblasts was quantified by real-time PCR. Data were expressed as ratio between mtDNA and nuclear DNA concentration. Results represent the mean of relative PCR ± SD of 3 independent experiments. Statistical analysis were performed by Student's t-test (two-sided).
X-linked inheritance of a frameshift mutation in TEX11.Figure 1Family tree of the azoospermic male patient WHT3759. The patient's brother was azoospermic with maturation arrest, and two maternal uncles were childless; DNA samples from these individuals could not be obtained. The patient's paternal uncle and all five paternal aunts each had two or three children (not shown). Complex frameshift mutation (patient WHT3759). The nucleotide sequence in exon 16 (in parentheses) changes from ACT(GATG) CCC to ACT(TTGGTA) CCC, resulting in a net insertion of two bases and generation of a KpnI restriction site (underlined). We confirmed heterozygosity of the mother (WHT4067) by KpnI digestion of PCR products (data not shown). Testicular maturation arrest in the patient (WHT3759). Testicular tissue obtained by biopsy was sectioned and stained with hematoxylin/eosin. Seminiferous tubules contained meiotic germ cells such as zygotene (ZS, arrowheads) and pachytene spermatocytes (PS, arrows), but no post-meiotic germ cells such as round spermatids were detectable. Scale bar, 25 μm.
Figure 7In wild type cells, SMCHD1 may remodel the telomeric chromatin which promotes ATM activation by the MRN complex (arrows). ATM activation is required for NHEJ at TRF2-depleted telomeres. In SMCHD1 knockout cells, ATM activation and DNA damage signaling is attenuated resulting in inefficient 3' overhang processing and impaired telomere end-to-end fusions. SMCHD1-loss and lack of ATM activation can be compensated for by ATR (not depicted). Only the subset of events and factors are depicted which are directly relevant to this paper.
Figure 5Figure 5. p53-dependent histone modification changes in response to DNA damage stress. HCT116p53-/- (left panels) or p53 +/+ (right panels) were treated with DMSO (black) or 50 µM etoposide (red) for 24 hrs and assayed by ChIP-qPCR at various locations relative to the TTAGGG repeats across the chromosome18qsubtelomere (panel A) or 13qsubtelomere (panel B) or for control regions (p21 promoter, GKN1/2, PARKIN) as indicated below. ChIP- for p53, H3K9Ac, H3K27Ac, γH2AX, or IgG control were quantified as % input. Error bars indicates SD and * indicates p value<.05 using student t-test comparing ChIP values in p53+/+ relative to p53-/- cells for specific primer pairs. (C) Dot blot analysis of telomere repeat or Alu repeat DNA from p53+/+ or p53-/- HCT116 cells treated with DMSO or etoposide (ETP) (as described in panel A), that were subject to ChIP with antibodies to IgG, p53, γH2AX, TRF2, or TRF1. Quantitation of three independent ChIP-assays for TRF1 and TRF2 is shown in the panel to the right. ( (E) ChIP-qPCR for TRF2 is shown for cells treated as in panel A using primers across the subtelomeres of 18q (left) or 13q (right).
Figure 6(A) Scheme of architecture and binding partners of the WAVE1 PWCA core domains (B) Overview of biochemical functions of WASP-family proteins in dendritic network assembly. (C) Two alternative models for the polymerase activity of WASP-family proteins. Note that the second model is not in agreement with thermodynamic laws (see Discussion).
Figure 4Transcript levels of (A) NIMIN1 and (B) PR1 in WT, anac032-1 and 35S:ANAC032 plants after 3 and 6 h of treatment with SA compared to their respective controls. (C), Expression of PDF1.2A in WT and anac032-1 plants after 3 and 6 h of treatment with SA compared to their respective controls. Transcript levels were measured using qRT-PCR and numbers on the y-axis indicate fold change (FCh; log2 basis). Means are shown ± SD (n = 3; 'n' represents independently performed experiments). Asterisks indicate a significant difference from wild type (p < 0.05; Student's t test).
LC3B and GATE‐16 overexpression differently affects phagophore appearance.Figure 5(A) Control HeLa cells and HeLa cells stably expressing GFP‐LC3B or GFP‐GATE‐16 were starved for 2 h in EBSS medium fixed and immunostained with anti‐Atg16 antibodies. Quantification of Atg16‐labelled puncta structures from three independent experiments is presented at the right panel. Arrowheads represent Atg‐16‐labelled puncta. Scale bar: 20 μm. (B) HeLa cells stably expressing GFP‐LC3B (left panel) or GFP‐GATE‐16 (middle panel) were starved for 2 h in EBSS medium. The cells were fixed and their cryo sections were immunolabelled as in ( Figure 4B). Quantification of the size of GFP‐labelled structures (right panel) from three independent experiments is presented. *P0.05, **P0.001.
Figure 3Cell size (A) of CD4+ T cells from P1 (red), P2 (blue), the mother (grey) and HD (black) stimulated with anti-CD3 (5 μg/ml) and anti-CD28 (10 μg/ml) in the presence or absence of 1 μM FK506 for 24 hours. (A) Representative histograms of FSC (left panel) and percentages of T cell blasts (defined as cells to the right of the dotted vertical line) analyzed by flow cytometry (right panel). proliferation (B) of CD4+ T cells from P1 (red), P2 (blue), the mother (grey) and HD (black) stimulated with anti-CD3 (5 μg/ml) and anti-CD28 (10 μg/ml) in the presence or absence of 1 μM FK506 for 24 hours. (B) Representative histograms of CFSE dilution (left panel) and percentages of proliferating cells (defined as cells to the left of the dotted vertical line) (right panel). Bar graphs in A and B are the mean ± SEM from two independent experiments. (C,D) Cytokine production by PBMC from P1, P2, the mother and an unrelated HD after stimulation with PMA (40 ng/ml) and ionomycin (500 ng/ml) for 4 hours. Cytokines were analyzed by flow cytometry following surface staining with antibodies against CD3, CD4 and CD45RO, permeabilization and intracellular cytokine staining for GM-CSF, IL-22 and IL-17A. Representative flow cytometry plots (C) and quantification of Th17 (GM-CSF, IL-22, IL-17A), Th1 (TNF-α, IFN-γ) and Th2 (IL-4) cytokines (D). Data represent the mean ± SEM from two independent experiments.
Modeling of multi‐pulse GF stimulation.Figure 6Deterministic modeling of responses to multi‐pulse EGF/NGF stimulation at different dosages. Integral of ERK activity in response to different pulse/pause regimes calculated from the ODE model for a duration of 60′, 90′ after GF stimulation. White dots indicate conditions, for which cell differentiation is evaluated.
Figure 2A. Triangular array region of FGF2 (PDB ID: 2FGF), FGF21core (β conformation) and FGF21core (loop conformation). The edges of the triangular formed by hairpins or missed (colored magenta) are indicated by solid or dashed lines, respectively. B. 2D 1H-15N Heteronuclear Single Quantum Correlation (HSQC) spectrum showing the two sets of assignments for residue Tyr22 to Glu50 (Labeled by one-letter amino acid code and sequence number). Labels for the loop conformation are colored red and marked with asterisks. C. 15N T1 and T2 relaxation times of the residues in β2-β3 region. The secondary structure of FGF21core (β conformation) is shown on the top. Error bars show the fitting SDs of ten different experiments. D. Secondary structure prediction of residues in β2-β3 region using TALOS+ program. The probability of β-strand and random coil of the residues are shown by histogram. E. 1H-1H slices of 15N-edited 3D NOESY-HSQC spectrum for β2-β3 residues. Residue codes are labeled as in B.
Figure 4A-C. Immunofluorescence images (A) and quantifications (B,C) of relative MAD1 and MAD2 kinetochore levels in indicated cells lines arrested in nocodazole and treated with/without AZ-3146 for 30 min to inhibit MPS1. In all kinetochore intensity graphs, each dot represents a cell, horizontal lines indicate the median and vertical bars show the 95% confidence interval. Note, when these vertical bars do not overlap the difference is considered statistically significant at a level of at least p<0.05 (see methods). All kinetochore intensity graphs display data that is relative to WT C11 controls in nocodazole, which are normalised to 1. The mean level of the normalised controls is indicated by the dotted lines. 30 cells from 3 experiments. Scale bars = 5µM. D. Duration of mitotic arrest in indicated cell lines arrested in nocodazole and then treated with 1.25 µM AZ-3146. Graph shows cumulative mean (±SEM) of 3 experiments, 50 cells per condition per experiment.
AID expression in pancreas promotes immune infiltration and cell death.Figure 5Hematoxylin-eosin (HE) staining of pancreas from aged (75-week-old) R26AID+/+p48CRE+/KI and R26AID+/KIp48CRE+/KI mice. Left: Representative HE staining showing an immune infiltrate in a R26AID+/KIp48CRE+/KI mouse. Scale bar: 200 μm. Right: Quantification of number of foci per mm2 of tissue. n = 13 (R26AID+/+p48CRE+/KI); 21 (R26AID+/KIp48CRE+/KI). **P = 0.0098. CD3 immunohistochemistry of pancreas from 75-week-old R26AID+/+p48CRE+/KI and R26AID+/KIp48CRE+/KI mice. Left: Representative image of a CD3 infiltrate in a R26AID+/KIp48CRE+/KI mouse. Scale bar: 100 μm. Right: Quantification of the number of CD3-positive cells per mm2 of tissue. n = 12 (R26AID+/+p48CRE+/KI); 13 (R26AID+/KIp48CRE+/KI). *P = 0.0149. Representative immunofluorescence staining of CD3 and CD8 in pancreas from 20-week-old R26AID+/+p48CRE+/KI and R26AID+/KIp48CRE+/KI mice. Scale bar: 20 μm. TNF-α expression. Total RNA was isolated from pancreas of aged (75-week-old) R26AID+/KIp48CRE+/KI and control mice, and TNF-α expression was quantified by qRT-PCR. Each dot represents an individual mouse. n = 8 (R26AID+/+p48CRE+/KI); 15 (R26AID+/KIp48CRE+/KI). *P = 0.0392. Cell death detection. Pancreas from 75-week-old R26AID+/+p48CRE+/KI and R26AID+/KIp48CRE+/KI mice were stained with anti-caspase-3. Left: Representative staining from an R26AID+/+p48CRE+/KI mouse (top) and an R26AID+/KIp48CRE+/KI mouse (bottom). Scale bar: 50 μm. Right: Quantification of number of cells per mm2 of tissue. Each dot represents an individual mouse. n = 9 (R26AID+/+p48CRE+/KI); 11 (R26AID+/KIp48CRE+/KI). P = 0.0545.
in vitro and EMRE peptide competition in cells.Figure 4A The binding of fluorescently (GFP) labelled MICU1 and mutants to EMRE peptide shown by MST in the presence of EGTA. B The binding of fluorescently (GFP) labelled MICU1 and mutants to EMRE peptide were analysed with MST in the presence of Ca2+. C Comparison of binding of MICU1 and EMRE peptide in both conditions. D MICU1-MICU2 heterodimer interacts with the EMRE peptide at a 1:1 ratio. E Carbachol (CCh)-induced representative traces of mitochondrial calcium uptake in HEK cells treated with mt-EMRE and scramble peptides. Solid lines, mean; shaded regions, ± SEM, n = 3. F Resting mitochondrial Ca2+ levels in mt-EMRE and scramble peptides treated cells, evaluated via ratiometric imaging of the mitochondrial targeted mt-riG6m. Mean ± s.d., student t-test, **p < 0.01, n = 3. Data information: Error bars for some points are too small to be visible. G Mean mitochondrial Ca2+ peaks response to 100 μM CCh. Mean ± s.d., student t-test, ***p ≤ 0.001, n = 3. Data information: Error bars for some points are too small to be visible. H Representative [Ca2+]c clearance traces of U2OS cells treated with PBS and mt-EMRE peptide. I Quantification of mitochondria Ca2+ uptake rate in response to 100 μM histamine. Mean ± s.d., student t-test, n.s., not significant, n = 4. Data information: Error bars for some points are too small to be visible.
Loss of ATG13 results in increased ATG9A recruitment to stalled clusters of p62/SQSTM1.Figure 6(A) Live cell imaging of HCT116 ATG13 WT and HCT116 ATG13 KO cells stably expressing mRuby2-ATG9A and EGFP-p62/SQSTM1. Representative montages show mRuby2-ATG9A recruitment to EGFP-p62/SQSTM1 puncta (Scale bar=5 µm). (B) Confocal images of Ubiquitin colocalization with p62/SQSTM1 in HCT116 ATG13 WT and HCT116 ATG13 KO cells. Cells were grown in full DMEM media, fixed, labelled with antibodies for Ubiquitin and p62/SQSTM1 and imaged (Scale bar=10 µm) (left). Quantification of Ubiquitin colocalization with p62/SQSTM1 (right). Mean ± SEM, n=1 independent experiment with 30 technical replicates. (C) Modified pulse chase experiment. HEK293T ATG13 WT and HEK293T ATG13 KO cells stably expressing HA ATG9A-BirA* were pulse labeled with biotin for 24 hours, media was replaced with full DMEM and streptavidin pulldown was performed at indicated time points. Quantification of normalized p62 infrared signal. Mean ± SEM, n=3 (biological replicates). Significance measured using Student's t test (bottom). ns=p>0.05, *=p≤0.05, **=p≤0.01, ***=p≤0.001, ****= p≤0.0001.
Ago1x can load miRNAs onto target mRNA (A) Linear regression analysis of miRNAs co-immunoprecipiated with Ago1 and those co-immunoprecipiated with Ago1x in HeLa cells.Figure 5Linear regression analysis of miRNAs co-immunoprecipiated with Ago1 and those co-immunoprecipiated with Ago1x in HeLa cells. Reads per million (RPM) values of 395 miRNAs, which were common to Ago1 and Ago1x are shown. qRT-PCR results showing the enrichment of seven miRNAs in Ago1x immunoprecipitate relative to that in Ago1 immunoprecipitate in HeLa cells. Bars show mean of three experiments ± S.E. RT-PCR results showing PTEN and MYC mRNAs co-immunoprecipitated with Ago1 or Ago1x in HeLa cells. Result of co-immunoprecipitation experiments demonstrating the interaction of Dicer protein with Ago1 and Ago1x in HeLa cells.
Figure 4Groups of BALB/c mice (n = 5) were inoculated three times, four weeks apart, with 20 µg of the postfusion F proteins indicated above each panel. One group (n = 5) was inoculated only with the CpG adjuvant, as indicated. Two other groups of mice (n = 8) were similarly inoculated with either F-415 or F-416, as indicated at top of the panels. A) Mouse sera, collected just before challenging with hRSV (A2 strain), were used in microneutralization assays against the viruses indicated at bottom. Statistical results are shown as in Fig. 3B. *P = 0.00085 and **P = 0.01986. Groups of BALB/c mice (n = 5) were inoculated three times, four weeks apart, with 20 µg of the postfusion F proteins indicated above each panel. One group (n = 5) was inoculated only with the CpG adjuvant, as indicated. Two other groups of mice (n = 8) were similarly inoculated with either F-415 or F-416, as indicated at top of the panels. B) Five days after virus inoculation, mice were sacrificed and the amount of virus in lung extracts was measured in a plaque assay. Mean virus titer for each group is indicated by short horizontal bar.
CRMP4 is elevated in ALS diseased spinal cord neurons (A, B) (A) Representative IHC images and (B) semi quantification of CRMP4 protein in human spinal cords (SC) cross sections from 2 control patients and 3 ALS patients.Figure 1(A, B) (A) Representative IHC images and (B) semi quantification of CRMP4 protein in human spinal cords (SC) cross sections from 2 control patients and 3 ALS patients. We analyzed total of 7 SC sections of controls and 14 SC sections of ALS patients, Data presented as mean ±SE. DAB: labeled CRMP4. Scale bar: left images 20 µm, right insets 10 µm. Mann-Whitney test ***p = 0.0003. (C) Representative images of P90 SC cross sections of SOD1G93A and WT mice. Red: denotes NeuN, Green: denotes CRMP4. Scale bar: 10 μm. (D) Quantification of the percentage of CRMP4 positive SC neurons in 3 WT VS. 3 SOD1G93A mice. We monitored CRMP4 expression in total of 108 cells in WT condition and 123 cells in SOD1G93A, an average of 36 or 41 cells in each repeat respectively. Student's t-test, n = 3, Data presented as mean ±SE, *p = 0.0161.