EMBO/sd-character-level-ner huggingface dataset:
Figure 2A. Fura2 340/380 time traces from M-CSF-differentiated macrophages derived from conditionally-immortalised macrophage precursor cell lines (M-MØP) of Plcg2P522 mice (P522, blue) and Plcg2R522 mice (R522, red). One set of cell lines, generated from male mice, is shown. Cells were exposed to 5µg/ml anti-FcγRII/III along with 20µM EGTA and 2µM Ionomycin as indicated. Data shows the mean±SD of 3 independent experiments analysed by two-way ANOVA with Sidak post-hoc tests (** = p<0.01; **** = p< 0.0001). B. Fura2 340/380 time traces from primary microglia derived from the cortex of Plcg2R522 mice (blue: P522) and Plcg2R522 mice (red: R522) with or without pre-exposure for 2 hours with Edelfosine (10µM). Cells were exposed to 5µg/ml anti-FcγRII/III along with EGTA and 2µM Ionomycin. Data shows the mean±SD of 3 independent experiments analysed by two-way ANOVA with Sidak post-hoc tests (** = p<0.01; *** = p<0.001; **** = p< 0.0001). C C. PLCG2R522 hIPSC-derived microglia show increased cytosolic Ca2+ influx in comparison to controls following activation of PLCγ2 using anti-CD32. Cytoplasmic Ca2+ increase following activation of PLCγ2 using anti-CD32. Three independent PLCG2R522CRISPR-engineered clones were examined. Data shown is mean±SD of 4 independent experiments and were analysed by one-way ANOVA with Tukey's multiple comparison test (* = p<0.05; ** = p<0.01).
Fusion profiles.Figure 5Fusion profiles. Cells exposed to 5-nm gold tracers for 20 (a and c) and 25 min (b) before fixation. Sections were picked up by MU and air-dried (c) or underwent immunolabeling with anti-SOD antibodies (a and b). (a) Fusion between a small VE (arrow) and an AVi. A cluster of two gold particles (arrowhead) is also seen at another location. (b) Fusion between an electron lucent MVE (arrow) and an AVd. (c) Fusion between an MVE (arrow) and an AVd. Bars, 200 nm.
Figure 2A Methyl-Cap-seq read density profiles of the 5' end of DAPK1, UNC5B, and NTN1in MDA-MB-231 (blue) and HMLER (green) cells. Red boxes represent the CpG islands (CGis); light gray boxes the regions analyzed by bisulfite-PCR-sequencing; dark gray boxes the regions analyzed by parallel sequencing of amplicons from bisulfite modified DNA; and black boxes the exons and UTR. Chromosome coordinates of each gene are given (black lines). B, C Gene expression was measured by Q-RT-PCR after 72 h for MDA-MB-231 (B) and HMLER cells (C) treated daily with DAC (10 µM). PBGD expression level was used as an internal control. Data are expressed as mean ± s.e.m. of at least 3 independent experiments. **** P<0.0001, two-tailed unpaired Student's t-test. D, E Measurement of DNA hypomethylation of the DAPK1 (D) and NTN1 (E) promoters after decitabine treatment of MDA-MB-231 and HMLER cells. Over 1960 sequences were analyzed per group in 2 independent experiments. **** P< 0.0001, two-way ANOVA and post-hoc Tukey-test.
Figure 3A. Apc1-loop500 is the hub for both Plx1 and PP2A-B56 phosphatase. CDK sites (T or S) and B56-binding site (green) on Apc1-loop500 (yellow bar) are shown. Bindings of Plx1 and B56 to Apc1-loop500 depend on phosphorylation of CDK sites. The PP2A-B56 associated on Apc1-loop500 may dephosphorylate CDK sites. B. Phosphorylation status of T532 and T539 in Apc1-loop500 varies depending on mutations in the B56-binding site. MBP-fused Apc1-loop500 WT or its derivatives (L557A/V560A and E562A) was incubated with anaphase extracts supplemented with non-degradable cyclin B at 23˚C for 1 h. The proteins were recovered by amylose beads, separated by SDS-PAGE and detected by immunoblotting with phospho-specific (pAb) or MBP antibody. C. Plx1-binding assay using MBP-fused Apc1-loop500 fragments with mutations in B56-binding site. The Plx1-binding to Apc1-loop500 WT or its derivatives (L557A/V560A or E562A) was analysed as described in Fig 1B. D. Quantification of (C). The bar graph is quantification of bound Plx1. The intensities of WT were arbitrarily set to 1.0. Error bars, SEM from three independent experiments. E. Binding assay using MBP fused Apc1-loop500 fragments and B56γ. MBP-fused Apc1-loop500 WT or its derivatives (T532A, T539A, T532A/T539A (2A) and S558A) was incubated with the 35S-labelled Flag-B56γ in anaphase extracts supplemented with non-degradable cyclin B at 23˚C for 1 h. The bound proteins were recovered by amylose beads, separated by SDS-PAGE and detected by autoradiography or Coomassie brilliant blue (CBB) staining. The recovery of WT (x1/2 and x1) was run as a standard. F. Quantification of (E). The bar graph is quantification of bound B56γ. The intensities of WT were arbitrarily set to 1.0. Error bars, SEM from three independent experiments. G. Summary of Plx1 and B56 binding to Apc1-loop500 derivatives. The abilities of Apc1-loop500 fragments carrying the indicated mutations are shown. The B56-bindings to ∆11 (deletion of 11 residues including B56 binding site) and 3A (T532A/T539A/S558A) were tested previously (Fujimitsu & Yamano, 2020).
Figure 2(A and B) Levels of IL-1β in supernatants from THP-1 cells that were subjected to knockdown as indicated and were treated with LPS, and then (A) treated with LLOMe or (B) starved in EBSS. (C) IL-1β in supernatants of primary human macrophages (MDM) subjected to knockdowns and sequentially treated with LPS and LLOMe. (D) Co-immunoprecipitation (co-IP) of flag-galectin-8 (Gal-8) and GFP-TRIM16 (T16) in HEK293T cells. (E) Co-IP analysis of endogenous galectin-8 and TRIM16 in protein complexes from LLOMe-treated THP-1 cells. (F) Confocal microscopy of THP-1 cells treated with LPS and LLOMe, and stained for TRIM16 and galectin-8. Line tracings correspond to arrowed dashed lines. Pearson correlation coefficient was calculated using SlideBook as described previously. Scale bars, 5 μm. (G) In vitro translated and radiolabeled [35S] myc-HA-TRIM16 was incubated with GST-galectin-8 in the presence (+) or absence (-) of flag-ULK1 and cold ATP, GST pulldowns were performed, and [35S] radiolabeled Myc-HA-TRIM16 in pulled-down material detected by PAGE and autoradiography. Amounts of GST fusion proteins are shown in coommassie brilliant blue (CBB)-stained gels. Data, means  SEM; n≥5, except for immunoblot quantifications where n≥3. *P < 0.05, †P ≥ 0.05 (t test or ANOVA).
Figure 4Lysates from T47D stimulated or not with FGF10, EGF or TGFα for different time periods (A) Lysates from T47D pre-treated or not with FGF10 for 40 min. and either stimulated or not with FGF10, EGF or TGFα for different time periods (B) Lysates from T47D stimulated or not with FGF10, EGF for different time periods or treated with PD173074 (C), MEK162 (D), AG1478 (E) before stimulation were immunoblotted with the indicated antibodies. F. Heatmap of the mRNA relative expression of the indicated ERK targets compared to control and quantified by qPCR. N=3. The minimum and maximum fold-induction is color-coded in white and burgundy, respectively. G, H Percentage of EdU incorporation in T47D pre-treated or not with FGF10 for 40 min. and stimulated or not with FGF10 or EGF (G) or incubated with MEK162, PD173074, or AG1478 before pre-treatment (H). N= 6. P value = <0.05 *, <0.01 **, <0.001*** (one-way ANOVA with Tukey test).
eRNA synthesis at super-enhancers and in large regulatory regions.Figure 4(A) Med1-defined enhancers ranked by Med1 ChIP-seq signal (Yan et al., 2013) in LoVo colorectal cancer cells. Super-enhancers (black rectangles) were determined as enhancer regions lying above the inflection point of the curve (B) Increased eRNA synthesis from super-enhancers. Boxplots comparing eRNA synthesis (TT-seq signal) at Med1-defined typical enhancers (n=2,111) and super-enhancers (n=130) in LoVo cells (analysis was restricted to intergenic enhancers Box limits are the first and third quartiles, the band inside the box is the median. The ends of the whiskers extend the box by 1.5 times the interquartile range. Notches represent 95% confidence intervals for the median values. Outliers not shown. P-value by two-sided Mann-Whitney U-test. (C) Super-enhancers are associated with the production of long eRNAs. Boxplots showing length of TT-seq-defined eRNAs originating from Med1-defined super-enhancers (n=552) or other enhancer regions (n=6,934). Median, hinges, whiskers and notches are shown as in (B). P-value by two-sided Mann-Whitney U-test. (D) Barplot showing the number of TT-seq enhancer regions per Med1-defined super-enhancer region in LoVo cells. Super-enhancers spanning a protein-coding TSS (n=32) are excluded from this comparison as regions TSS ± 1 kbp were excluded from TT-seq enhancer calling. (E) UCSC genome browser view of normalized TT-seq coverage on the plus and minus strand at the PHLDA1 locus (hg38; chr12:75,612,350-76,042,552; in the colorectal cancer cell line HCT 116. TT-seq coverage is cut at 250 (purple lines) to allow for better visualization. eRNAs are shown between the plus and minus strand. Aggregated transcription factor ChIP-seq peak regions (TF ChIP-seq) comprise ChIP-seq binding profiles of 19 TFs in HCT 116 cells from CISTROME The height of the signal indicates the number of different TFs binding at a particular region. Binding regions for selected TFs (TCF7l2, YY1, SP1) are shown below together with p300 and RNA polymerase II (Pol II). TT-seq enhancer regions are shown at the bottom together with a H3K27ac-defined super-enhancer annotation, which was downloaded from SEdb 2019). At date of publication no Med1 ChIP-seq data was available for HCT 116.
The S.Figure 8. The S. typhimurium-induced macrophage death does not require caspase activity. BMDPM were infected with wild-type S. typhimurium or the TTSS-defective invG mutant in the presence or absence of 50 μM of the pan-caspase inhibitor Z-VAD, and cell death was measured as indicated in the legend to Fig. 1.
Figure 1A. Expression instability of the chicken BU-1 locus as a reporter for replication impediments formed by structure-forming DNA sequences. The leading strand of a replication fork entering the locus from the 3" end encounters a DNA sequence with structure-forming potential located 3.5 kb downstream of the transcription start site. In wild type cells this is a G4 motif, which is replaced by (GAA) n repeats in this study. Under conditions in which polymerase stalling is prolonged, e.g. loss of G4 processing enzymes or G4 stabili or repriming is defectiv the persistence of a putative ssDNA gap leads to a zone of interrupted histone recycling and loss of parental histone modifications. If this loss of modifications involves a control region of the gene, e.g. the promoter, it can result in a change in expression B. Flow cytometry for Bu-1a expression in wild type cells with (GAA) n tracts of different length knocked into the BU-1A locus (in blue). DT40 cells are heterozygous and carry one BU-1A and one BU-1B allele. All experiments introducing repeats into BU-1A are carried out in cells in which the +3.5 G4 has been deleted from both A and B alleles, to avoid transvection between the allele Black outline: positive control (wild type cells); red outline: negative control (cells carrying a genetic disruption of BU-1) C, D. Bu-1a fluctuation analysis of wildtype and primpol cells in which the endogenous +3.5 G4 has been deleted (ΔG4) or with (GAA)10 and (GAA)20 sequence orientated such that it is replicated as the leading (C) or lagging (D) strand template for a fork entering from the 3' end of the locus as shown in panel (A). At least two independent fluctuation analyses were performed. Circles represent the percentage of Bu-1a loss variants in at least 24 individual clones from these experiments, with mean ± SD reported. ****p < 0.0001, * p ≤ 0.05, ns = not significant; one-way ANOVA.
DNA methylation changes during neuronal differentiation of adult hippocampal neural precursor cells.Figure 1A, Adult hippocampal neural precursor cells (NPCs) were differentiated into neurons. DNA methylation patterns of NPCs and purified neurons were analyzed by Reduced Representation Bisulfite Sequencing (RRBS; n = 3 cultures per group). B, Volcano plots depicting CpG methylation changes between cell populations. Significantly differentially methylated CpGs with q < 0.05 and absolute methylation differences greater than 20 % are highlighted in violet (demethylated CpGs - Demeth) and red (de novo methylated CpGs - De novo). Percentages of differentially methylated CpGs among all CpGs covered by RRBS are indicated with the respective color. C, Differentially methylated CpGs were depleted at promoters and CpG islands but enriched at gene bodies and enhancers compared to all CpGs covered by RRBS. Stars indicate FDR adjusted p < 0.05 (linear regression). D, Genes with known role in adult hippocampal neurogenesis as annotated in the Mammalian Adult Neurogenesis Gene Ontology (MANGO; 359 annotated genes) were significantly enriched among de novo methylated genes (494 genes; p-values are from hypergeometric test). Numbers of overlapping genes are indicated at the respective bars. E-F, Candidate gene regions with neurogenesis-associated de novo methylation (E) and demethylation (F). Depicted are mean methylation levels per group at individual CpGs within the specified genomic regions. Stars indicate significantly differentially methylated CpGs with q < 0.05 (logistic regression with multiple testing correction using SLIM method) and methylation difference greater than 20 %.
Figure 2(A) Plot from flow cytometry analysis of neutrophils in forepaw skin from 10-wk-old WT and RDEB mice. (B) Bar graph of flow cytometry analyses as in A of neutrophils in forepaw skin of newborn, 4-wk-old and 10-wk-old WT and RDEB mice. Plotted as fold increase compared to mean of age-matched WT paws; replicates per age = 5-6. ( Data information: Individual data points, mean ± S.E.M are shown. The data were analyzed by one-way ANOVA with Tukey's correction P values < 0.05 are considered significant. (C) Plot from flow cytometry analysis of CD38+ and Egr2+ macrophages in forepaw skin from 10-wk-old WT and RDEB mice. (D) Bar graph of flow cytometry analyses as in C of inflammatory macrophages (CD38+Egr2-) or tissue-repair macrophages (CD38-Egr2+) macrophages in forepaw skin of newborn, 4-wk-old and 10-wk-old WT and RDEB mice. Plotted as fold increase compared to mean of age-matched WT paws; replicates per age; n = 5-6 (CD38+Egr2-); n = 4-5 (CD38-Egr2+). ( Data information: Individual data points, mean ± S.E.M are shown. The data were analyzed by one-way ANOVA with Tukey's correction P values < 0.05 are considered significant. (E) Plots of MHC II staining intensity in neutrophils (top) and in macrophages (bottom) from flow cytometry analysis of forepaws from 10-wk-old WT and RDEB mice. (F-G) The mean fluorescence intensity (MFI) of MHC II staining on (F) neutrophils (n = 3-4 biological replicates) and (G) macrophages (n = 6-7 biological replicates). Data information: Individual data points, mean ± S.E.M are shown. The data were analyzed by one-way ANOVA with Tukey's correction P values < 0.05 are considered significant. (H) Systemic inflammation in RDEB mice. The percentage abundance of neutrophils and monocytes in blood of 4-wk-old WT and RDEB mice, (n = 5-7 mice). Data information: Individual data points, mean ± S.E.M are shown. The data were analyzed by one-way ANOVA with unpaired t-test P values < 0.05 are considered significant. (I) Analyses of percentage CD3ε+ positive CD45+ cells (T-cells) in paws, and percentage CD4 or CD8 single positive T cells, (n = 3-4 biological replicates) Data information: Individual data points, mean ± S.E.M are shown. The data were analyzed by one-way ANOVA with Tukey's correction P values < 0.05 are considered significant. (J) Staining of back skin from 10-wk-old WT and RDEB mice for IgG (red). Keratinocytes are stained with syndecan-1 (green) and 4′,6-diamidino-2-phenylindole (DAPI) (blue) was used to visualize nuclei, scale bar = 100 μm.
Figure 3(A) Schematic of (CGG) 90-EGFP construct and experimental outline for rough eye phenotype screening. (B) Quantitation of GMR-GAL4 driven uas-(CGG) 90-EGFP eye phenotype with candidate modifiers (n > 30 flies/ genotype). siNTC = siRNA against a non-targeting control gene (mCherry). Different siRNA lines for the same target gene are numbered (#1 and #2). Error bars represent mean +/- SD. Data information: For eye scoring, target siRNA lines were compared to non-targeting control siRNA lines using a two-tailed student's t test with Welch's correction for multiple comparisons. ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001. Human orthologs of fly genes are used for labeling. (C) Representative photographs of fly eyes expressing either GMR-GAL4 driver alone or with uas-(CGG) 90-EGFP construct, with fly SRSF1 (dSF2) and SRSF2 (dSC35) knockdown or disruptions (insertion). (D) Representative photographs of fly eyes and quantitation (below) of rough eye scores with fly SRSF1 overexpression (dSF2 OE); n=20-32/genotype. Error bars represent mean +/- SD. Data information: For eye scoring, target siRNA lines were compared to non-targeting control siRNA lines using a two-tailed student's t test with Welch's correction for multiple comparisons. ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001. Human orthologs of fly genes are used for labeling. (E) Representative photographs of fly eyes expressing GMR-GAL4 driven (GGGGCC) 28-EGFP with indicated uas-siRNAs against fly SRSF genes in comparison with non-targeting control (NTC) siRNA against LUC/luciferase. (F) Quantitation of (GGGGCC) 28-EGFP rough eye phenotype with SRSF modifiers (n > 30 flies/ genotype). Error bars represent mean +/- SD. Data information: For eye scoring, target siRNA lines were compared to non-targeting control siRNA lines using a two-tailed student's t test with Welch's correction for multiple comparisons. ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001. Human orthologs of fly genes are used for labeling. (G) Representative photographs of fly eyes expressing GMR-GAL4 driven (GGGGCC) 28-EGFP at 29˚C along with the quantifications of necrosis and eye width. n=28-30/genotype. Error bars represent mean +/- SD. (H-I) Survival assays of flies expressing (CGG) 90-EGFP under Tub5-GS (H) and ELAV-GS (I) drivers with control or SRSF1 siRNAs. Expression of (CGG) 90-EGFP was initiated with addition of drug starting 1 day post eclosion and continued through experiment (Log-rank Mantel-Cox test; n=98-101/genotype for Tub-GS and n=120-141/genotype for ELAV-GS flies) ∗p < 0.05, ∗∗p < 0.01. (J) Survival assays of (GGGGCC) 28-EGFP expressing fly under Tub5-GS driver (Log-rank Mantel-Cox test; n=71-93/genotype) with control or SRSF1 siRNAs. ∗∗p < 0.01.
Acute depletion of Chk1 in the hematopoietic system leads to cell loss in vitro and retention of CHK1 expression in vivo.Figure 8Three independent Chk1fl/fl mT/mG (coloured) and mT/mG (grey) Hoxb8-FL progenitor cell lines were transduced with a retroviral construct carrying Cre (pMIP-CRE-IRES-PURO) and monitored over time for GFP-expression. Shown here are three technical replicates per genotype. Additionally, Chk1fl/fl mT/mG Hoxb8-FL progenitor cells were FACS-sorted for GFP+ and dsRED+ cells 48h after transduction with the Cre-construct. Sorted cells were processed for western blotting using the indicated antibodies. Chk1fl/fl Hoxb8-FL progenitor cells were transduced with a retroviral construct carrying Cre and GFP (pMIG-CRE-IRES-GFP). Cells were FACS-sorted 72h after transduction with the retroviral construct and processed for western blotting using the indicated antibodies. Percentage of GFP+ cells indicative for CRE expression and recombination in the peripheral blood after tamoxifen administration by gavage (5 x 2 mg, 200 μl/mouse; daily). Bars represent means ± S.E.M. (Day 0 N=9/10, WT/Chk1fl/fl, Day 7 N=8/7, Day 14 N=3/3). Quantification of Chk1-del PCR-product enrichment in DNA from GFP+ vs. Tomato+ bone marrow cells sorted on day 8 after first TAM administration. Transgenic Cre levels were quantified in parallel for reference. Bars represent means ± S.E.M. from n=5 mT/mG Vav-CreERT2 Chk1fl/fl mice. Percentage of living LK or LSK-cells and (F) percentage of GFP+ LK or LSK-cells found in the bone marrow of mT/mG Vav-CreERT2 (WT) and mT/mG Vav-CreERT2 Chk1fl/fl (Chk1fl/fl) mice on day 8 or 15 post first TAM administration. Bars in (E)/(F) represent means ± S.E.M. (Day 8 N=8/8, WT/Chk1fl/fl, Day 15 N=7/8). GFP+ (CRE) and Tomato+ (CRE-) total bone marrow cells were sorted on day 8 after first TAM-administration and processed for Western Blot using the indicated antibodies. Sorted cells from three animals - mT/mG Vav-CreERT2 (WT) and mT/mG Vav-CreERT2 Chk1fl/fl (Chk1fl/fl) - were pooled per lane. Thymocytes of the indicated genotypes were sorted based on GFP+ and Tomato+ on day 15 after the first TAM administration for western analysis using the indicated antibodies.
Loss of Miro1/2 reduces short-range peroxisomal displacements.Figure 4A) Snapshots and tracks of pxDsRed signal in WT, Miro1KO, Miro2KO and DKO MEFs following live imaging at 1.5 seconds per frame for two minutes. B) Quantification of median net displacement of individual peroxisomes (pxDsRed signal) for WT, Miro1KO, Miro2KO and DKO MEFs (n=42 cells over six independent experiments. Two different MEFs lines were used for each genotype). Data information: For multiple comparisons in B) a one-way ANOVA with Newman-Keuls post-hoc test was used to test for statistical significance. C) Still images every 30 seconds of ER and peroxisomes (ER-DsRed pseudo-coloured to green and pxGFP pseudo-coloured to magenta, respectively) in WT and DKO MEFs taken from two-minute movies by live-cell spinning-disk microscopy. Arrows track individual peroxisomes associated with an ER tubule. Scale bar is 5 μm. D) Quantification of the relative ER displacement over a two-minute movie using ER-DsRed signal in WT and DKO MEFs (n=20 cells per condition over three independent experiments). Data information: Student's t-test was used to calculate statistical significance. * and *** represent p<0.05 and p<0.001, respectively. Data are represented as mean ± SEM. E) Median net displacement of peroxisomes in WT and DKO MEFs. n=20 cells per condition over three independent experiments. F) Median net displacement of peroxisomes in DKO MEFs with and without myc-tagged variant 4 of Miro1 (mycv4) (n=12 cells per condition over three independent experiments). Data information: Student's t-test was used to calculate statistical significance. * and *** represent p<0.05 and p<0.001, respectively. Data are represented as mean ± SEM.
Figure 1A. Schematic of the p53 mouse model which generates L2-Cre;Trp53+/+;R26lox-Stop-lox-YFP, L2-Cre;Trp53-/-;R26lox-Stop-lox-YFP and L2-Cre;Trp53R172H/- ;R26lox-Stop-lox-YFP mice. Mice were treated with 4NQO in the drinking water for 16 weeks followed by normal water. Esophageal epithelial cells were isolated and cell lines were generated. B. Cell lines generated from WT, null, and mutant p53 esophagi were assessed by Western blot analysis for p53 protein expression. Relative intensity densitometry of Rab11-FIP1 results are shown at the bottom. C. Organoids generated from WT, mutant, and null p53 cell lines. Scale bar: 100μm. D. Quantification of the portion of organoids exhibiting a solid, intermediate or stratified morphology (> 37 organoids from 2-3 cell lines per group). E-G. Cells were subcutaneously injected in the lower flanks of nude mice (n= 4 flanks/cell line. WT p53 2 cell lines, Mutant p53-3 cell lines, p53 null-3 cell lines). E. The number of tumors formed in each group was quantified. F. Tumor growth was measured at the indicated time points post-implantation. Graph represents total tumor size per group. G. Average tumor weight following dissection (n=2 biological replicates for WT p53 cell lines, n=3 biological replicates for mutant p53 cell lines, n=3 biological replicates for p53 null cell lines, n=4 technical replicates injected per cell line, calculated as average tumor weight per cell line). Error bars represent ±S.E.M. *p=0.02 Mutant p53 vs WT p53, *p=0.02 Mutant p53 vs Null p53 (One-way Anova).