EMBO/sd-character-level-ner huggingface dataset:
Figure 1A, Schematic drawing of the EGFP-based SSA reporter, EGFP-SSA. The EGFP coding sequence is split into two fragments, which share a 315 bp overlapping homology (repeat) and are separated by a 2,035 bp DNA sequence. An I-SceI cleavage sequence is inserted in the intervening sequence, 9 bp away from the 3' repeat. The EGFP open reading frame is restored after DSB-induced RMD of the two EGFP fragments. B, RMD assays performed after DSBs have been generated by sgRNAs/Cas9. The distance between the sgRNA/Cas9 cleavage sites and the repeat sequences is indicated. U2OS cells carrying a single copy of the EGFP-SSA reporter (3427-17) were transfected with sgRNA/Cas9 plasmids, and the percentage of GFP-positive cells was determined (left). U2OS cells transfected with the indicated reporters EGFP-SSA-17/6 bp, EGFP-SSA-0.3 kb, EGFP-SSA-0.6 kb and EGFP-SSA-1.0 kb were pooled as a population after selection of the drug marker on the reporters, followed by sgRNA/Cas9 cleavage (right). C, RMD is strongly induced when a DSB is generated close to one repeat. The U2OS (EGFP-SSA-3427-17) cell line carrying a single copy of the reporter was transfected with sgRNA/Cas9 plasmids as indicated, and the percentage of GFP-positive cells was determined. D, Models of RMD mediated by SSA (RMD/SSA) or BIR (RMD/BIR). When a DSB is created in the middle of two repeats, the DNA ends of both sides are resected, generating 3' ssDNA overhangs, until the repeat sequences on opposite sides are both exposed. Then ssDNA of the repeat sequences anneal, resulting in a product with internal sequences deleted. When a DSB is generated close to one repeat, ssDNA formed in one repeat first can invade the other repeat still in duplex using BIR, which can also give rise to a deletion product identical to the SSA product after cleavage of D-loop. Pink arrow: putative structure-specific endonucleases to cleave non-homologous tails; blue arrow: putative structure-specific endonucleases to cleave the D-loop. Determination of RMD frequency in U2OS (EGFP-SSA-3427-17) cells when POLD3 is depleted by lentiviral expression of shRNA. Cells infected with a lentiviral empty vector were used as control. The knockdown level of the indicated proteins was shown by Western blot, using KU70 as the loading control. Determination of RMD frequency in U2OS (EGFP-SSA-3427-17) cells when RAD52 is depleted by lentiviral expression of shRNA. Cells infected with a lentiviral empty vector were used as control. The knockdown level of the indicated proteins was shown by Western blot, using KU70 as the loading control. G, Determination of RMD frequency in U2OS (EGFP-SSA-3427-17) cells when RAD51 is depleted by lentiviral expression of shRNA. Cells infected with a lentiviral empty vector were used as control. The knockdown level of the indicated proteins was shown by Western blot, using KU70 as the loading control.
A Non-cullin neddylated species accumulate after oxidative stress in WT U2OS cells with the most highly induced species detected at 25 kDa (as indicated by the arrow).Figure 3Non-cullin neddylated species accumulate after oxidative stress in WT U2OS cells with the most highly induced species detected at 25 kDa (as indicated by the arrow). WT U2OS cells were treated with the indicated concentration of H2O2 for 30 minutes and then lysed in LDS sample loading buffer and processed for Western blot analysis. (Left) The 25-kDa neddylated species induced by oxidative stress increases with time in WT U2OS cells. U2OS WT or NEDP1 KO cells were treated with 600 μM H2O2 for the indicated time and then lysed in LDS sample loading buffer and processed for Western blot analysis. (Right) The same 25 kDa NEDD8 species, which is strongly induced in WT cells, is present in untreated NEDP1 KO cells and does not further increase after oxidative stress induced by H2O2 (as indicated by the arrow). U2OS NEDP1 KO cells are resistant to the PARP-1 inducer, H2O2. WT and NEDP1 U2OS cells were plated in 96-well plates and after 24 hours were treated with the indicated concentration of H2O2. They were assessed for viability 24 hours later by the CellTiter-Glo assay. Graphs represent the mean ± S.E.M. of the percent survival compared to untreated cells. NEDP1 KO cells are re-sensitized to H2O2, following re-expression of NEDP1 by transient transfection. U2OS cells were plated in 96 well plates and reversed transfected with NEDP1 or an empty vector. 48 hours after plating, cells were challenged with the indicated amount of H2O2 and cell viability was assessed 24 hours later by the CellTiter-Glo assay. Graphs represent the mean ± S.E.M. of the percent survival compared to untreated cells. Neddylation of cullins is decreased in NEDP1 KO cells and is rescued by re-expression of NEDP1 by transient transfection. Western blot analysis from HEK 293 WT, NEDP1 KO cells and NEDP1 KO cells rescued with transient transfection of NEDP1 reveals that upon NEDP1 re-expression, the intensity of the NEDD8 reactive bands is reduced and the levels of free NEDD8 are increased. NEDP1 re-expression also increased the neddylation of each Cullin. The neddylated band of each Cullin is denoted with a star. Quantification and graph of the mean ± S.E.M. of the percentage neddylation of each Cullin in (E). Deletion of Nedp1 does not lead to induction Nrf2 response genes. RNA was harvested from WT and NEDP1 KO U2OS cells, reversed transcribed to cDNA, and analysed by qPCR for Nrf2 and NQO1 expression. Deletion of Nedp1 but not of Cand1 in HEK 293 cells results in resistance to cell death from H2O2. WT, NEDP1 KO, and CAND1 KO cells were plated in 96 wells plates and treated with the indicated amount of H2O2. 24 hours after treatment, cell viability was measured using the CellTiter-Glo assay. Graphs represent the mean ± S.E.M. of the percent survival compared to untreated cells.
Figure 1A, Genome browser images of ChIP-seq for SAGA proteins before and after induction of ER stress at ER stress response genes CHOP, ERP70, and GRP78. ATF4/Xbp1s binding sites are from publicly available datasets (GEO accessions GSE69304 and GSE49952). B, Heat map of USP22, GCN5, and ATXN7L3 association at the TSS for 29 genes whose promoters undergo significant recruitment of USP22 following induction of ER stress. C, Mean ChIP-seq signal for USP22, GCN5, and ATXN7L3 surrounding gene promoters from B. Nonparametric Mann-Whitney analysis of 7 bins (grey shade) surrounding maximal peak in Thaps treated conditions are reported. D‑F, Linear regression analysis of SAGA member recruitment to target gene promoters following induction of ER stress.
Figure 9A and B, ChIP-qPCR assay of HDA15's (A) and NF-YC9's (B) residence at target promoters in presence or absence of TOC1 and PRR5. GFP-HDA15 and GFP-NF-YC9 driven by 35S promoter were transiently expressed in protoplasts isolated from Col-0, toc1 single and prr5 toc1 double mutant grown in SD condition. Results were normalized by % input of PIL1 coding region (PIL1 CDS). Data from at least 3 independent experiments were averaged, error bars indicate SEM. Asterisks indicate significant differences (* p<0.05, ** p<0.001, *** p<0.0001, Student's t test). C and D, Dual luciferase assay of the repression activity of GFP-TOC1 and GFP-5X on promoter of AT5G02580 (C) and CDF5 (D) in protoplasts of toc1 and hda15toc1 (h15t1) mutants. AT5G02580:LUC, CDF5:LUC reporters and the effectors (empty GFP, GFP-TOC1, and GFP-5X) were co-transfected to toc1 and hda15 toc1 protoplasts with 35S:Renilla-LUC as internal control. Data are means ± SEM (n = 5 biological trials) relative to empty GFP which is set as 1. Different letters indicate statistically significant differences (student's t test, p<0.05).
Figure 3A+B. Images showing representative HEK S1/S2-/- cells co expressing YFP-STIM1 (green) or either STIM1-mCherry or STIM1A-mCherry (red) and merged images before (A) and after (B) stimulation with TG. C. Images showing representative HEK293 cells expressing Orai1-GFP (green = donor molecule) or either STIM1-mCherry (upper panel = acceptor molecule) or STIM1A-mCherry (lower panel = acceptor molecule) after stimulation with TG. Also shown is the overlay of the two channels, as well as the FRET signal. D. Co-localization analysis of cells from (C). For each condition 9 cells were analyzed using Mander's overlap coefficients (M1 and M2) S1 M1=0.68, M2=0.71; S1A M1=0.55, M2=0.65. Data information: Data was obtained from three independent transfections is shown as single values with corresponding median. Scale bars, 10 µm. E. Quantification of FRET signal measured in C (STIM1: 0.28, n=37, STIM1A: 0.26, n=39). Data information: Data was obtained from three independent transfections and, is shown as single values with corresponding median. Scale bars, 10 µm. F. Distribution of cluster sizes in HEK293 cells transfected with STIM1-mCherry (black, n = 2644) or STIM1A-mCherry (red, n = 4754) at 0.2 µm2 intervals in %. G. Violin blot of the cluster shown in (F), shown as median, first and third quartile. ns: not significant with Mann Whitney test. D
Identification of a novel BCL‐2‐interacting partner, NAF‐1, at the ER.Figure 1(A) BH3‐only BIK displaces a subset of BCL‐2 complexes at the ER. Purified LM from H1299 HA‐BCL‐2b5 cells either mock infected or infected with Ad‐BIKb5 was subjected to cross‐linking with BMH and visualized by immunoblot. Asterisks (*) denote BCL‐2 cross‐linked products, which are displaced by BIK. Bold arrow denotes region containing cross‐linked NAF‐1/BCL‐2. (B) Amino‐acid sequence of NAF‐1. Peptide sequences identified by mass spectrometry are indicated by a double underline. The TM domain is indicated by a bold single underline. Cys residues in the cytosolic domain are denoted with asterisks (*). The CDGSH domain is highlighted in grey, and the predicted 2Fe‐2S coordinating Cys‐3‐His‐1 aa are marked in bold. A downwards arrow head (∨) represents the site in which a Flag epitope was inserted. A double‐lined arrow (⇓) indicates the Glu residue that when mutated (E37Q) causes WFS2. Predicted phosphorylation sites found using the PhosphoMotif Finder from the Human Protein Reference Database (HPRD) are indicated in bold within a box; β‐adrenergic receptor kinase substrate motif (aa 25-29), PKA and PKC kinase substrate motifs (aa 32-34 and 67-69), and casein kinase II substrate motif (aa 34-37). (C) Deduced membrane topology of NAF‐1. Grey rectangle indicates CDGSH domain. (D) Endogenous NAF‐1 cross‐links with BCL‐2 at the ER. LM purified from H1299 neo and HA‐BCL‐2b5 cells were subjected to cross‐linking by BMH and immunoprecipitation with anti‐NAF‐1 antibody. Precipitates were analysed by immunoblot with anti‐BCL‐2. (E) NAF‐1 is displaced from BCL‐2 by BH3‐only BIK. H1299 HA‐BCL‐2b5 cells were either mock infected or infected with Ad‐BIK. Purified LM was treated as in (D).
HOTAIR expression perturbs LSD1 activity at enhancers.Figure 5Venn diagram representing intersections of active enhancer regions in Epi cells. Diagram representing putative active enhancers identified by LILY in each cell line and shared between low- or high-migrating groups respectively. Metagene profiling of H3K27ac, LSD1 and H3K4me2 over IgG within the 5 kb window of enhancers in Epi cells expressing none (CTR) or different HOTAIR variants (HOT, HOTΔP and HOTΔL). bp stands for base pairs.
ActivinB promotes cell proliferation in Group 3 MB cell lines.Figure 3D458 cell lines were treated with PBS (vehicle, black) or with ActivinB (green). Immunoblot of phosphorylated Smad2 (P-Smad2), total Smad2 and β-actin in response to ActivinB stimulation for 24 hours. Quantification of P-Smad2 (P-S2) to β-actin are shown on right panels. Bars represent the mean ± SD. Number of replicates is n≥3. D458 cell lines were treated with PBS (vehicle, black) or with ActivinB (green). Growth curve experiments showing cell proliferation upon ActivinB treatment. The p-values were determined by unpaired t-test and two-way ANOVA *p < 0.05, **p < 0.01, ***p < 0.001. D458 cell lines were treated with PBS (vehicle, black) or with ActivinB (green). Cell cycle analysis by FACS measuring BrdU incorporation and 7AAD labelling at 48 hours upon ActivinB stimulation. The percentage of cells in the different phases of the cell cycle is represented (G0/G1, S and G2/M phases). Bars represent the mean ± SD. Number of replicates is n≥3. D458 cell lines were treated with PBS (vehicle, black) or with ActivinB (green). Percentage of apoptotic cells measured by FACS analysis of cleaved caspase3 48h hours after treatment with ActivinB. Bars represent the mean ± SD. Number of replicates is n≥3. D283 cell lines were treated with PBS (vehicle, black) or with ActivinB (green). Immunoblot of phosphorylated Smad2 (P-Smad2), total Smad2 and β-actin in response to ActivinB stimulation for 24 hours. Quantification of P-Smad2 (P-S2) to β-actin are shown on right panels. Bars represent the mean ± SD. Number of replicates is n≥3. D283 cell lines were treated with PBS (vehicle, black) or with ActivinB (green). Growth curve experiments showing cell proliferation upon ActivinB treatment. The p-values were determined by unpaired t-test and two-way ANOVA for F. *p < 0.05, **p < 0.01, ***p < 0.001. D283 cell lines were treated with PBS (vehicle, black) or with ActivinB (green). Cell cycle analysis by FACS measuring BrdU incorporation and 7AAD labelling at 48 hours upon ActivinB stimulation. The percentage of cells in the different phases of the cell cycle is represented (G0/G1, S and G2/M phases). Bars represent the mean ± SD. Number of replicates is n≥3. D283 cell lines were treated with PBS (vehicle, black) or with ActivinB (green). Percentage of apoptotic cells measured by FACS analysis of cleaved caspase3 48h hours after treatment with ActivinB. Bars represent the mean ± SD. Number of replicates is n≥3.
EGFR and c-Fos co-expression in human OS negatively correlates with patient survival.Figure 6Representative images of OS biopsies stained with antibodies against EGFR and c‑Fos. Scale bars: 50µm Kaplan-Meier survival curve comparing the survival of patients with EGFR and c-Fos double positive osteosarcomas against patients without co-expression of both proteins. (n=52) Gene expression correlation analysis of several cancer-associated RTKs with FOS in human OS (data analyzed from publicly available dataset E-GEOD-39058)
a) Insolubilization of Keap1 in Atg7-deficient hepatocytes.figf4(a) Insolubilization of Keap1 in Atg7-deficient hepatocytes. Liver homogenates from Atg7F/F:Mx1 mice on various days after injection of poly(I)•poly(C) were separated into detergent-soluble and detergent-insoluble fractions with 0.5% Triton X-100. Each fraction was subjected to SDS-PAGE and analysed by immunoblotting with the indicated antibodies. The data displayed are representative of three separate experiments. (b) Immunoblot analysis of Atg7-deficient (Atg7F/F:Mx1; Atg7F/F shown here as F/F) and Atg7 p62-deficient (Atg7F/F:Mx1:p62−/−) livers. Liver homogenates from mice of the stated genotypes at 12 days after injection of poly(I)•poly(C) were separated into detergent-soluble and detergent-insoluble fractions. Each fraction was subjected to SDS-PAGE and analysed by immunoblotting with the indicated antibodies. Atg7F/F mice in which Atg7 is efficiently expressed at a level similar to that in the wild-type mice were used as control. Data shown are representative of three separate experiments. Uncropped images of blots are shown in Supplementary Information, Fig. S11. (c) Quantitative real-time PCR analyses of Nqo1 and Gstm1 in mouse livers. Total RNAs were prepared from livers of the indicated genotypes at 12 days after injection of poly(I)•poly(C). Values were normalized to the amount of mRNA in Atg7F/F liver. Data are means ± s.d. for three experiments. (d) Immunofluorescence analysis of the cellular localization of p62 and Keap1. Liver sections from mice of the indicated genotypes at 28 days after injection of poly(I)•poly(C) were immunostained with anti-Keap1 (top) and anti-p62 (middle) antibodies. Bottom: merged images of Keap1 (green) and p62 (red). Each inset in the Atg7-deficient liver panels is a magnified image of the boxed region. Scale bars, 20 μm.
Figure 6A, High-throughput proteomic analysis of Mediator tail subunits MED16 and MED24 following depletion of USP22, with significance measured by Student's t-test. Specific ubiquitylated lysines detected are highlighted in red text. B, HCT116 cells were treated for 2 hrs with 100 nM Thapsigargin in the presence or absence of shRNA targeting USP22. Cells were harvested, fractionated, and fractions were subjected to immunoblotting with the indicated antibodies. C, In vitro deubiquitylation UbiTest of endogenously ubiquitylated MED24. HCT116 cells were treated with MG132 in the presence or absence of shRNA targeting USP22. Lysates were generated using buffer containing protease cocktail inhibitor, pan-DUB inhibitor PR619, and the JAMM protease inhibitor o-phenanthroline. Ubiquitylated proteins were purified on ubiquitin binding resin and eluates were either undigested (lanes 1 and 2) or digested with USP2 to strip polyubiquitin (lanes 3 and 4) and reduce target proteins to unit length. Digestion reactions were subjected to immunoblotting with the indicated antibodies.
Figure 6A. 13C-serine tracing into the metabolic network of iRBC. Black arrows indicate reactions predicted from genomic reconstruction, whereas green arrows indicate reactions that are not predicted but best match the stable-isotope labelling pattern observed. Proposed reactions are annotated below. Metabolites in grey were not observed via mass spectrometry and green indicates a metabolite observed but not predicted. Highlighted in orange is the glycine cleavage system, which is predicted to be active but has not been verified. B. One-carbon folate metabolism in P. falciparum. The parasite encodes all the enzymes necessary for a complete cytosolic pathway but only appears to possess a single enzyme-serine hydroxymethyltransferase-in the mitochondrion. Highlighted in white are the conventional enzymes and reactions of one-carbon metabolism in the mitochondria of other eukaryotes. C. HA-tagged SHMT-M detection via immunofluorescence microscopy. DAPI was used to visualise the nucleus, and MitoTracker CMXros used to illuminate the mitochondrion. Anti-HA signal partially colocalises with the mitochondrion. D. Addition of 100 nM rapamycin and 2.5 mM glucosamine for three growth cycles (+) leads to depletion of the HA-tagged SHMT-M. E. Depletion of SHMT-M leads to significant changes in intracellular levels of many metabolites in iRBC. m/z features are plotted as the log2 ratio of treated/untreated intensities and the Benjamini-Hochberg corrected P values across six biological replicates plotted as -log10(P). F. Pathway enrichment analysis of the m/z features identified as significantly different when SHMT-M is depleted. Depicted are the Gene Set Enrichment Analysis (GSEA) P values and Mummichog P values generated via Metaboanalyst 3.0, with the combined P value from each pathway enrichment approach represented by color.
Figure 8(A-D) Effect of rad52∆, rad51∆ (A), rad57∆ (B) and rad54∆ (C) on PCNA ubiquitylation in cells synchronized in G1 and released into fresh medium in the presence of 0.025% MMS. Cell cycle progression was followed by cell sorting analysis and western blot against Clb2. Pgk1 was used as loading control. The amounts of mono- and polyubiquitylated PCNA were normalized to the total amount of PCNA. For each time point, rad mutant values were relativized to those of the wild type, taken as 1. The mean and SEM of 3 independent experiments are shown (D). An asterisk indicates a mean significantly different than 1, according to a One-sample t-test. (E) Model for Rad52-mediated tolerance during DDT. The recombination factors Rad52, Rad51 and Rad57 promote efficient Rad6/Rad18 binding to chromatin under unperturbed conditions. In response to blocking lesions that impair replication fork advance the Rad6/Rad18 complex is mobilized to ssDNA lesions where, together with Mms2/Ubc13/Rad5, ubiquitylates PCNA and promotes TS. The ssDNA lesions that escape from this error-free pathway during S phase accumulate in G2/M, where they are filled in by two additional, less accurate mechanisms regulated by Rad52: UbPCNA-independent HR (recombinogenic) and TLS (mutagenic). Apart from facilitating Rad6/Rad18 chromatin recruitment, Rad52 plays an additional uncharacterized Rad51/Rad57-independent role in TLS.
Figure 4Type XVII collagen (COL17, arrowheads indicate the hair bulge) and laminin β1 (LAMB1) labeling in WT dorsal skin sections (P1). Scale bar: 100 μm. Blistered samples of Col17a1-/- mouse dorsal skin at P1. ITGA6 (indicated by arrowheads) and COL4 (arrows) labeling (B). L332 staining (C, arrows). Scale bar: 100 μm. H&E staining of blistered skin from Col17a1-/- mice at P1. HFs detached from the dermis in Col17a1-/- skin are indicated by arrowheads. Scale bar: 500 μm. Whole-mount AP staining of the blister roof epidermis from Col17a1-/- mice (right) and littermate controls (left) at P1. Scale bar: 500 μm. H&E (P2, left), ITGA6 (P2, middle) and LOR staining (P4, right) of Col17a1-/- mice (top) and littermate controls (bottom) . The regenerated epidermis is indicated by arrowheads. Scale bar: 200 μm. BrdU labeling of Col17a1-/- skin at P2. Scale bar: 100 μm. (Bottom) Quantification of BrdU-positive cells in the epidermis surrounding blisters (n= 3 (control) and 4 (Col17a1-/-) biological replicates). The data are shown as the mean ± SE. Student's t-test. NS, no significance. Lineage tracing of K14CreER:R26R-mCherry:Col17a1-/- at P4. Scale bar: 100 μm. Quantification of mCherry-positive cells in the regenerated epidermis (n=3 biological replicates). The data from individual mice are connected by lines. *0.01 test.
PR-SET7-deficient hepatocytes die via necrosis in a cell division-dependent manner.Figure 3A Electronmicroscopic images of cells containing enlarged nuclei in P120 PR-SET7ΔHepA livers and normal hepatocytes in control littermates (WT). White arrows indicate: (Nuc) nuclei; (ER) endoplasmic reticulum; (Mit) mitochondrium; (PM) plasma membrane; (a) swollen mitochondria; (b) swollen endoplasmic reticulum; (c) disorganized endoplasmic reticulum; (d) disrupted plasma membrane; and (e) disorganized pieces of endoplasmic reticulum encircling mitochondria to form autophagosomes. B Immunohistological staining of liver sections from P120 PR-SET7ΔHepA mice and control littermates (WT) with H4K20Me1 and H4K20Me3 antibodies. Normal (Area-A), Necrotic (Area-B) and Regenerative (Area-C) hepatocyte-containing areas are indicated. Arrows show the loss of H4K20Me1 signal from the large necrotic nuclei. C-F Partial (2/3rd) hepatectomy was performed in 45-day-old PR-SET7ΔHepA mice and control littermates (WT). Two weeks later, at postnatal day 60 (P60) liver sections were stained with hematoxylin and eosin (C) or γH2AX (D) or H4K20Me1 (E) or HNF4 and F4/80 (F) antibodies.
Comparison between Sir4 and Dbf4 H‑BRCT domains and phospho-peptide binding to Dbf4 H-BRCT A) Superposition of Sir4 (orange) and Dbf4 (PDB 3QBZ, cyan) H‑BRCT domains highlighting the structural conservation of the Sir4 phospho-epitope binding region in Dbf4.Figure 6Superposition of Sir4 (orange) and Dbf4 (PDB 3QBZ, cyan) H‑BRCT domains highlighting the structural conservation of the Sir4 phospho-epitope binding region in Dbf4. The only change is the Arg1066 (Sir4 H‑BRCT) replacement to Lys200 (Dbf4 H‑BRCT), highlighted by an arrow, while all other residues are conserved. MST analysis of the binding interactions between Dbf4 H‑BRCT and Cy5-labeled phospho (filled circles) and non-phospho peptides (empty circles) from Esc1 and Ubp10. KD = equilibrium dissociation constant, which could not be determined (n.d.) for non-phospho peptides. ΔFnorm [0/00] represents the change in fluorescence during thermophoresis normalized to the initial fluorescence. Data are the mean ± SEM from ≥3 independent measurements.