EMBO/sd-character-level-ner huggingface dataset:
Microscopy-based siRNA screen of human E2 ubiquitin-conjugating enzymes identifies UBE2QL1 to be required for ubiquitination of damaged lysosomes.Figure 1A Robust immunofluorescence detection of ubiquitination on LAMP1-positive compartments with pan-ubiquitin FK2 or K48 chain-specific antibodies. HeLa cells were treated with 250 µM LLOMe or EtOH (untreated) for 3 h, fixed and stained with indicated antibodies. Confocal microscopy was followed by automated image analysis. Graphs represent data from three independent experiments with ≥ 50 cells per condition (mean ± SD). Scale bar: 20 µm. B Screen overview. C Primary screen using pools of four single siRNAs targeting 37 human E2 enzymes. HeLa cells were seeded in 384-well plates, transfected with a siRNA library in quadruplicates for 72 h. Cells were LLOMe-treated and processed for imaging Plates were imaged automatically with a spinning disk confocal microscope. Automated image analysis was done using the Acapella software and the percentage of ubiquitinated lysosomes was determined. Robust z-scores were calculated for LLOMe-treated samples for each depletion against the values of the whole plate. Samples exceeding the threshold of -2 of robust z-score (highlighted in red) were considered for the secondary screen. D Selected candidates were re-screened with the same work pipeline but with the four single siRNAs of pools of each candidate from the primary screen. Candidates exceeding the threshold in both stainings are highlighted in red. E Damage-induced translocation of UBE2QL1 to lysosomes. The three best candidates (from D) were transiently expressed with N-terminal HA-tag in HeLa cells. Cells were treated with LLOMe or EtOH (untreated) for 3 h, fixed and stained for HA and LAMP1 as indicated and imaged by confocal laser scanning microscopy. Scale bar: 10 µm. F Automated quantification of (E). Shown are Pearson correlation coefficients (P.C.C) of HA and LAMP1 signals. Graph represents data from three independent experiments with ≥ 30 cells per condition (mean ± SD). ****P<0.0001 (One-way-ANOVA with Bonferroni's multiple comparison test). G Western blot detection of endogenous UBE2QL1 and depletion efficiency. Cells were transfected with control siRNA (Ctrl), two UBE2QL1 siRNAs from the screen (#2 or #4) or an additional siRNA (#5), and lysates probed with an antibody to UBE2QL1 as indicated. GAPDH was probed as loading control.
Flagellar sub-complexes in L.Figure 2Sub-tomogram average of the IM sub-complex constituting the C- ring, MS-ring and export apparatus. Schematic representation of the sub-tomogram average shown in (A) highlighting the different parts of the complex. Sub-tomogram average of the motors of fully-assembled flagella. Slices through electron cryo-tomograms showing neighboring PL and IM sub-complexes. Dashed-yellow circles highlight the IM sub-complex while dashed-blue arrows highlight the PL sub-complex. Dashed-red lines mark the border between two images used to make a composite image when the PL and IM sub-complexes were found at different levels in the tomogram. Schematic representation of an IM sub-complex in the vicinity of a PL sub-complex. Slices through electron cryo-tomograms of L. pneumophila highlighting the presence of a fully-assembled basal body lacking the hook and the filament. Schematic representation of the basal bodies in (L and M). Central slice through an electron cryo-tomogram of a lysed cell. The dashed-yellow circle highlights the flagellar motor. Enlarged view of the same slice shown in (O). The absence of the C-ring and the export apparatus is highlighted by the dashed-orange arrow. Schematic representation of the complex found in (P).
Figure 2A. Structure of 5-azacytidine and formation of a covalent RNA methyltransferase adduct. B. HEK293 cells expressing NSUN3-HisPrcFLAG (NSUN3) or the HisPrcFLAG-tag alone (FLAG) were either not crosslinked (-), UV crosslinked (UV) or treated with 5-azacytidine (5-AzaC). The protein-RNA complexes were affinity purified and the bound RNA was trimmed, end-labelled with 32P phosphate and ligated to linkers. Protein-RNA complexes were separated by SDS-PAGE, transferred to nitrocellulose and exposed to an X-ray film. C-E. The UV or 5-AzaC crosslinking and analysis of cDNA (CRAC) experiments with NSUN3-HisPrcFLAG (D-E) or the FLAG control (C) samples were treated as described in B. The RNA was isolated from the nitrocellulose membrane-bound protein-RNA complexes and converted into cDNA for sequence library production and Illumina deep sequencing. Pie charts present different RNA classes and the relative distribution of Illumina sequence reads that were obtained after mapping of the reads on the human genome. Bar graphs below indicate the distribution of mitochondrial (mt-)tRNA, mt-rRNA and mt-mRNA sequence reads among the reads mapped to the mitochondrial genome. Abbreviations: tRNA - transfer RNA, snRNA - small nuclearRNA, snoRNA - small nucleolarRNA, rRNA - ribosomal RNA, mtRNA - mitochondrial encoded RNA, miscRNA - miscellaneous RNA, miRNA - micro RNA, lncRNA - long non-coding RNA. F. Relative distribution of mitochondrial tRNA sequence reads obtained from the CRAC experiments using UV or 5-AzaC crosslinking with cells expressing the NSUN3-HisPrcFLAG (NSUN3) protein or control cells (FLAG). Only mt-tRNAs that were represented by more than 5% of all mt-tRNAs reads are labelled. G. 5-AzaC crosslinking was performed and RNA associated with wildtype NSUN3, the catalytically inactive NSUN3 mutant (C265A) or the FLAG tag alone was isolated as described in (B). The RNA analysed by northern blot using probes against the mt-tRNAMet, mt-tRNAPro and mt-tRNAGlu. Inputs (0.1%) are shown on the left and eluates (50%) on the right.
Figure 12(A) In wild-type cells, the disaccharide pentapeptide subunit linked to the undecaprenyl lipid transporter (Lipid II) is translocated to the outer leaflet of the cytoplasmic membrane by MurJ and polymerized by the glycosyltransferase and D,D-transpeptidase activities of the PBP2-RodA complex. MepM is essential and sufficient for insertion of new material in the PG net, although this endopeptidase can be replaced by overproduction of MepH, MepS, or PBP7. (B) Inhibition of PBP2 by β-lactams leads to the accumulation of uncross-linked glycan chains that are cleaved by the Slt70 lytic transglycosylase. This limits the supply of glycan chains for cross-linking by the YcbB L,D-transpeptidase. Under this condition, MepM or overproduction of MepS is required for insertion of new glycan strands. Production of YcbB leads to a requirement for an additional endopeptidase, MepK. This function of MepK can be bypassed by overproduction of MepS. (C) Deletion of the sltY gene encoding lytic transglycosylase Slt70 prevents digestion of uncross-linked glycan chains leading to a full supply of neo-synthesized glycan chains to YcbB and improved expression of β-lactam resistance. IM, inner membrane; OM, outer membrane; CRO, ceftriaxone.
Rag GTPases and mTORC1 regulate the function of MITF.Figure 8.(A) Multisequence alignment of the first 30 amino acids of TFEB with MITF isoforms 1, 2, 3, and 7. The green arrows indicate the residues identified as essential for the interaction between TFEB and Rag proteins. Blue letters are to indicate conserved amino acid homology between the different proteins. (B-D) ARPE-19 cells were infected with adenovirus expressing MITF1-FLAG. 16 h later, cells were incubated with DMSO or 250 nM Torin-1 for 1 h and analyzed by immunoblotting (C) or immunofluorescence (B and D) with anti-FLAG antibodies. The region within the dotted box is magnified in the insets. Bars: (B and D, main images) 10 µm; (D, insets) 5 µm. (E) ARPE-19 cells were incubated with 250 nM Torin-1 for 1 h. Cells were then lysed and analyzed by immunoblotting with antibodies against MITF. (F) HEK-293T cells were incubated in medium containing DMSO or Torin-1 for 1 h. Cells were lysed, and nuclei and membrane plus cytosol fractions were obtained by low speed centrifugation. Proteins from the different fractions were subjected to immunoblotting with antibodies against MITF, Lamp1, and Histone H3. (G) ARPE-19 cells expressing active (RagBGTP/RagDGDP) or inactive (RagBGDP/RagDGTP) Rag heterodimers were immunoprecipitated with the anti-FLAG antibody and immunoblotted with antibodies against FLAG and GST (used to detect MITF and Rag proteins, respectively). (H) HEK-293T cells expressing active (RagBGTP/RagDGDP) or inactive (RagBGDP/RagDGTP) Rag heterodimers were pulled down using glutathione-Sepharose beads and immunoblotted with antibodies against GST and MITF (used to detect Rag proteins and endogenous MITF, respectively). IP, immunoprecipitation.
CUL3 and KLHL3 are present in mouse and human aorta, and CUL3WT/Δ403–459 mice undergo aortic vascular remodelling.Figure 7Western blot of tunica media-intimathoracic aorta lysates from mice culled after a minimum 4-h fast. Following exsanguination after surgery, mouse tissues were rapidly harvested and the tunica adventitia removed before storage, and later, the samples were homogenised, clarified and quantified prior to SDS-PAGE. Western blot analysis confirmed the expression of KLHL3 and CUL3. Similar to the kidney, the aorta of CUL3WT/Δ403-459 showed slightly lower levels of CUL3 compared to CUL3WT without any change in KLHL3 levels. Fresh frozen thoracic aorta tissues from donor cadavers were homogenised, clarified and quantified prior to SDS-PAGE. Western blot analysis confirmed the expression of KLHL3 and CUL3 in normal healthy human aorta. Representative maximum-intensity z projections of immunofluorescence stained thoracic aorta sections showing the distribution of CUL3 and KLHL3 between CUL3WT/Δ403-459 and CUL3WT mice at a minimum 4-h fasting baseline (n = 4 per genotype). CUL3 and KLHL3 localisation is comparable between genotypes. The highest levels were detected in the vascular smooth muscle cells and endothelium, with a minimal expression in the perivascular adipose tissue of the adventitia. Scale bar, 50 μm. Representative immunohistochemistry staining of KLHL3 and CUL3 in humanthoracic aorta sections (n = 6). Similar to the mouse staining seen in (C), vascular smooth muscle cells in the tunica media of the aortic wall are positive for KLHL3 and CUL3. Scale bar, 50 μm. Morphometric analysis of thoracic aortae reveals vascular remodelling in CUL3WT/Δ403-459 mice. There is an increase of ˜21% in the vessel wall intima-media thickness (demarcated by arrows) of CUL3WT/Δ403-459 compared to CUL3WT mice (***P = 0.0003). However, there is no change in the number of elastin laminae (P = 0.1458) and therefore no increase in the number of medial muscle layers between genotypes. Two-tailed unpaired Student's t-test; data are mean ± SEM.
Figure 2A GST pulldown binding assay of in vitro transcribed/translated 35S-Myc-CALCOCO1 with recombinant GST-tagged ATG8 family proteins. GST and GST fusion proteins were visualized by coomassie brilliant blue staining (bottom panel), and the co-precipitated Myc-CALCOCO1 was detected by autoradiography (upper panel). The numbers below the AR represent % binding in the shown AR . B GST pulldown assay of transiently transfected Myc-CALCOCO1 from HEK293 cell extracts with recombinant GST-tagged ATG8 family proteins. GST and GST fusions were visualized by Ponceau S staining (bottom panel), and co-precipitated Myc-CALCOCO1 detected by immunoblotting with anti-Myc antibody (upper panel). C CALCOCO1 deletion constructs used to map the ATG8 interactions. The red X indicate a point mutation or deletion of the LIR motif. Constructs with no or very weak interaction are indicated in orange. GST pulldown assays of indicated in vitro transcribed/translated 35S-Myc-CALCOCO1 constructs with indicated recombinant GST-tagged ATG8 family proteins. Precipitated GST and GST fusions and co-precipitated Myc-CALCOCO1 constructs were analyzed as in A. GST pulldown assays of indicated in vitro transcribed/translated 35S-Myc-CALCOCO1 constructs with indicated recombinant GST-tagged ATG8 family proteins. Precipitated GST and GST fusions and co-precipitated Myc-CALCOCO1 constructs were analyzed as in A. GST pulldown assays of indicated in vitro transcribed/translated 35S-Myc-CALCOCO1 constructs with indicated recombinant GST-tagged ATG8 family proteins. Precipitated GST and GST fusions and co-precipitated Myc-CALCOCO1 constructs were analyzed as in A. GST pulldown assays of indicated in vitro transcribed/translated 35S-Myc-CALCOCO1 constructs with indicated recombinant GST-tagged ATG8 family proteins. Precipitated GST and GST fusions and co-precipitated Myc-CALCOCO1 constructs were analyzed as in A. GST pulldown assays of indicated in vitro transcribed/translated 35S-Myc-CALCOCO1 constructs with indicated recombinant GST-tagged ATG8 family proteins. Precipitated GST and GST fusions and co-precipitated Myc-CALCOCO1 constructs were analyzed as in A. I GST pulldown assay of endogenous GABARAP from HEK293 cell extracts with recombinant GST-tagged CALCOCO1 constructs. GST and GST fusions were visualized as in A, and co-precipitated GABARAP with anti-GABARAP antibody (upper panel).
Frequencies of ommatidia with different numbers of rhabdomeres are shown at 3 days after eclosion in progeny of flies expressing mutant huntingtin exon 1 (gmrQ120) crossed to either a control stock (w1118; W) or to Dhc64C (dynein heavy chain mutant; Dhc; a) or Roblk (dynein light chain mutant; Robl; b) flies.figf4Frequencies of ommatidia with different numbers of rhabdomeres are shown at 3 days after eclosion in progeny of flies expressing mutant huntingtin exon 1 (gmrQ120) crossed to either a control stock (w1118; W) or to Dhc64C (dynein heavy chain mutant; Dhc; a) or Roblk (dynein light chain mutant; Robl; b) flies. P ≤ 0.0001 (Mann-Whitney U test). (c) Ommatidia from the progeny of flies expressing mutant huntingtin exon 1 (Q120) crossed with either control stock (w1118; W) or with Dhc64C (Dhc) flies. All fly experiments were repeated three times independently with similar significance levels. One representative experiment is shown.
Hypothetical model depicting the effect of SPPL2c overexpression on vesicular trafficking.Figure 7A) Membrane-tethered glycosyltransferases (represented here by GnT-V in green) and members of the SNARE family (dark and light blue representing t- and v-SNAREs respectively) are synthesized and inserted in the ER. The glycosyltransferases are transported to the Golgi with the aid of the SNAREs via vesicle transport. Within different compartments of the Golgi they mature and either meet SPPL3 (red) that can shed and liberate their active site, or they catalyze protein glycosylation (glycoprotein depicted in purple). B) Upon expression of SPPL2c (orange) in the ER, selective SNARE proteins are proteolytically processed. C) Cleavage of SNARE proteins impairs vesicle fusion at the cis-Golgi and amongst others, immature glycosyltransferases accumulate in the ER, while the amount of glycosyltransferases in the Golgi decreases. This leads to a decrease in protein glycosylation and at the same time, SPPL3 (red) probably decreases, most likely to preserve the small amount of glycoproteins that have reached the Golgi.
Scl binds to enhancers that have been primed for activation in mesodermDistribution of Scl binding sites relative to TSS in Scl‐bound activated or repressed genes shows that majority of Scl binding sites locate away from TSS.Figure 2A Distribution of Scl binding sites relative to TSS in Scl‐bound activated or repressed genes shows that majority of Scl binding sites locate away from TSS. Chi‐square test was used to assess differences in the distribution. B Percent of tissue‐specific enhancers validated by LacZ reporter mice (Vista enhancer browser) that overlap with SclMES binding sites shows that Scl binds to experimentally verified heart enhancers more often compared to enhancers from other tissues. C Percent of SclMES binding sites that overlap with heart enhancers in E11.5 embryos defined by p300 binding (Blow et al, ) is higher than overlap with enhancers from other tissues. D SclMES binding sites overlap with experimentally verified heart‐specific enhancers upstream of Myocardin and Nkx2.5 genes and with hematopoietic enhancers within Runx1 and Gata2 genes. E Percent of enhancers from different cardiac developmental stages identified by H3K4me1 (enhancers) and H3K4me1 combined with H3k27ac (active enhancers) (Wamstad et al, ) that overlap with SclMES binding sites. F Average H3K4me1 (left) and H3K27ac (right) profiles around all 4,393 Scl binding sites show tissue‐specific enrichment in Flk1+mesoderm (MES), cardiac precursors (CP) and cardiomyocytes (CM) but not in ESC (mouseES cells) mouse embryonic fibroblasts (MEF) and embryonic limbs (LIMB). G Correlation analysis of H3K4me1 (left) and H3K27ac (right) levels between WT and SclKOMES and ES cells and MES (Wamstad et al, ) around 4,393 Scl binding sites shows that establishment of these marks occurs independently of Scl. Scl, H3K4me1 and H3K27ac ChIP‐seq tracks show comparable levels of H3K4me1 and H3K27ac in WT and SclKO mesoderm around cardiac (Gata6, Tbx5) and hematopoietic (Eto2, Gfi1b) genes.
ATF7IP represses the SETDB1-regulated exogenous provirus reporter and ERVs in mESCs A Atf7ip KO cells show increased expression of SETDB1-regulated ERVs and the provirus reporter, MSCV-GFP (EV1B), as evidenced by RT-qPCR analysis.Figure 1A Atf7ip KO cells show increased expression of SETDB1-regulated ERVs and the provirus reporter, MSCV-GFP as evidenced by RT-qPCR analysis. RNA expression was normalized to Hprt expression and is shown relative to the level in WT cells. Data are mean ± SD; n=3, technical replicates. B Atf7ip KO mESCs (TT#2-12) show decreased H3K9me3 at the LTR of the SETDB1-regulated reporter and the ERVs, as evidenced by Native ChIP followed by qPCR analysis. Gapdh was used as a negative control. Data are mean ± SEM; n=5, independent experiments. *P < 0.05 and **P < 0.01 by paired t-test. C SETDB1 cellular amount is mostly maintained in Atf7ip KO mESCs. Endogenous SETDB1-ATF7IP interaction is validated by anti-ATF7IP antibody co-IP experiment with anti-ATF7IP antibody.
Figure 4(a) Prediction of drug response in terms of survival: The Y-axis displays the hazard ratio of patients as a function of upregulation of predicted rescuers (b) Analyzing drug response in terms of tumor size reduction (RECIST criteria): the predicted DU-SR rescuers of drugs are differentially over-expressed in non-responding tumors. The Y-axis denotes the fraction of the predicted drug-specific rescuers that are over-expressed (out of all predicted rescuers of that drug) in tumors of responders (red) and non-responders (blue). Significant results are marked by stars (Wilcoxon Rank sum P < 0.05, aggregate Wilcoxon rank sum is P < 2.2E-16
Figure 2Targets from each mode of regulation (cf. Fig 1E) were selected for validation by Nanostring. Shown is a scatterplot of Nanostring quantification of polysome-associated mRNA vs. cytosolic mRNA log2 fold-change (shERα vs. shCtrl; n = 3). Genes are colored according to their identified mode of regulation (i.e. from Fig 1E). Confidence ellipses (based on Nanostring data, level 0.7) are overlaid for each mode of regulation. Selected genes from each mode of regulation are indicated. Levels of indicated proteins from shERα and control BM67 cells were determined by western blotting with their quantifications. β-actin served as loading control. Graphs represent mean ± standard deviation (n = 3-5 as indicated). Protein expression between groups were compared using two-sided paired Student's t tests. Immunoblotting of BM67 cell lines extract and its quantitation following incubation with 100 µg/mL cycloheximide as indicated. Graphs represent mean ± standard deviation (n = 5).
Figure 1(A) The transcript level of solo LTR in the wild type and the epcr1 mutant. Error bars are standard deviations of three technical replicates. The experiment was independently performed three times and similar results were obtained. (B) The developmental phenotype of the epcr1 and epcr2 single mutants and the epcr1/2 double mutant relative to the wild type. Two-week-old seedlings grown on vertical MS medium plates are shown. (C) The transcript levels of solo LTR, SDC, AtSN1, AtGP1 and AtCOPIA28 in the wild type, epcr1 and epcr2 single mutants, epcr1/2 double mutant, and two individual EPCR1 transgenic lines in the epcr1/2 double mutant background. Error bars are standard deviations of three biological replicates.
Figure 6Quantifications of neutral comet assay showing the level of DSBs under indicated siRNA-depleted conditions, before and after IR (4 Gy). (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001; two-way ANOVA, n=3, mean +SEM). Colony forming assay (CFA) showing the IR sensitivity of HeLa cells under siRNA-mediated p97 or ATX3-depleted conditions. (n=3, mean ±SD).   CFA showing the IR sensitivity of HeLa-WT cells compared to HeLa∆ATX3 cells. (n=3, mean ±SD).   CFA showing the rescue of cell survival in IR treated, ATX3-depleted (siRNA), HeLa cells, after DOX-inducible expression of ATX3-WT but not with ATX3-C14A or ATX3-UIM* variant. (ns P>0.05, **P<0.01, ****P<0.0001; two-way ANOVA, n=3, mean ±SD).   CFA showing the rescue of cell survival in IR treated, ATX3-depleted (siRNA), HeLa cells, after DOX-inducible expression of ATX3-WT but not with ATX3-VBM variant. (ns P>0.05, *P<0.05, ***P<0.001, ****P<0.0001; two-way ANOVA, n=3, mean ±SD).   Representative IF micrographs in U2OS cells showing IRIF formation of GFP-RNF8-WT, 53BP1 and γH2AX (first panel) and GFP-RNF8-∆RING, 53BP1 and γH2AX (second panel) under indicated siRNA-depleted or p97-inhibited (CB5083) conditions. (Scale bar; 10µm).   Quantification of (F) for GFP-RNF8-WT. Graph represents the percentage of cells with increased GFP-RNF8-WT foci (white bars) or percentage of cells with >5 53BP1 foci (grey bars). (***P<0.001, ****P<0.0001; two-way ANOVA, n=2, mean +SEM, at least 100 nuclei per condition and experiment).   Quantification of (F) for GFP-RNF8-∆RING. Graph represents the percentage of cells with increased GFP-RNF8-∆RING foci (white bars) or percentage of cells with >5 53BP1 foci (grey bars). (ns P>0.05, *P<0.01, **P<0.01, ****P<0.0001; two-way ANOVA, n=2, mean +SEM, at least 100 nuclei per condition and experiment).   Representative IF images and quantification showing endogenous K63-Ub and γ-H2AX at UV-A micro-laser-induced DNA damage tracks in U2OS cells under indicated siRNA-depleted conditions. (Scale bar; 10µm).   Quantification of (I). Graph represents the average intensity of the K63-Ub (ns P>0.05, **P<0.01, ***P<0.001; unpaired t-test, n=2, mean +SEM, more than 50 nuclei were analysed).