EMBO/sd-character-level-ner huggingface dataset:
UBE2QL1 recruitment coincides with the delayed K48-linked ubiquitination response.Figure 3A HeLa cells expressing UBE2QL1-HA were fixed at indicated time points after LLOMe treatment and processed for confocal microscopy. Cells were stained with antibodies specific for LAMP1 (not shown), and combinations of HA with K48 chains, K63 chains or p62 as indicated. Note the delayed co-emergence of K48 chains and UBE2QL1 vesicles, compared to the earlier appearance of K63 chains and p62. Arrows indicate colocalizing or non-colocalizing vesicles. Scale bars: 10 µm, 2 µm for inlays. B Automated quantification of (A). Shown are the Pearson correlation coefficients (P.C.C.) representing colocalization of UBE2QL1-HA, K48 chains, K63 chains or p62 with LAMP1-positive vesicles. Graphs represent data from three independent experiments with ≥ 30 cells per condition (mean ± SD). C Automated quantification of (A). P.C.C.'s representing colocalization of K48 chains, K63 chains or p62 with UBE2QL1-HA in cells treated with LLOMe or vehicle alone (untreated) for 180 min. Graphs represent data from three independent experiments with ≥ 30 cells per condition (mean ± SD).
a deficiency.Figure 5(A) Analysis of Sμ-Sα recombination junctions in the genomic DNA isolated from siControl- and siPhf5a-transfected CH12F3-2A cells stimulated by CIT for 48 h. The values are presented as mean ± sd (n=3), Statistical significance was evaluated in reference to siControl by two-tailed unpaired Student's t-test (*p ≤ 0.05, ** p ≤0.01, *** p ≤0.001). (B) The proportion of insertions versus microhomology regions of various lengths in the Sμ-Sα junctions presented as a pie chart. The number of clones analyzed from control and Phf5a-depleted samples are indicated at the center. (C) Top: schematic diagram of the position of the ChIP assay PCR products specific to the S region. Bottom: ChIP assay of the indicated DNA repair proteins using CIT-stimulated CH12F3-2A cells transfected with siControl or siPhf5a. The values are presented as mean ± sd (n=3). Statistical significance was evaluated in reference to siControl by two-tailed unpaired Student's t-test (*p ≤ 0.05, ** p ≤0.01, *** p ≤0.001).
Figure 7Confocal images of γ-irradiated GFP-NLS-, GFP-hcGAS-expressing HEK293 cells stained for RAD51 (red) with or without γ-irradiation. Scale bar: 10 μm. Schematics of the D-loop formation assay, including pre-incubation of template dsDNA with cGAS-cat (i), or with cGAS-cat being added after RAD51 was bound to dsDNA (ii). Pre-incubation of dsDNA with mcGAS-cat prevents D-loop formation by human RAD51, but does not affect the RAD1 activity once RAD51 filaments are bound to dsDNA. The percentage of D-loop formed in each reaction (left) was graphed as the average of triplicates ± SD. Schematics of the D-loop assay. Pre-incubation of template dsDNA with hcGAScat blocks subsequent D-loop formation. The percentage of D-loop formation (below) was graphed as the average of triplicates±SD.
Figure 6A) Expression analysis by qRT-PCR (mean ± SD) of the indicated intestinal epithelial cell markers regulated by the Wnt and Notch signaling pathways that are involved in stem cell homeostasis, regionalization, and Goblet cell differentiation (n=3). B) Genomic snapshots of the indicated genomic loci for SUZ12 occupancy and H3K27me3 deposition in intestinal crypts of wild type mice (n=3). C) Expression analysis by qRT-PCR (mean ± SD) of the Goblet cell markers (Atoh1, Gfi1, Spdef), early secretory precursor cells (Dll1), NOTCH targets (Hes1, Olfm4) and intestinal stem cells (Lgr5, Rnf43) in wild type intestinal crypts purified 38hrs after treatment with the NOTCH inhibitor DBZ. DMSO served as vehicle negative control (n=4). D) ChIP analysis of Atoh1 and Gfi1 loci using H3K27me3 specific antibody. The same crypts described in C were used. H3K27me3 levels were normalized on Histone H3 density (percentage of H3 signal). Graphs show mean ± SD. E) Graphic model highlighting the role of PRC2 in restricting secretory lineage differentiation and cell cycle checkpoint activation in TA cells.
Effects of Rubicon on autophagosomal maturation and HCV RNA replication in HCV replicon cells.Fig 6(A) Increase of Rubicon, UVRAG, p62 and LC3-II in HCV subgenomic RNA replicon cells. Actin served as the loading control. Numbers under Rubicon and UVRAG indicate the protein levels of Rubicon and UVRAG in replicon cells relative to their levels in control Huh7 cells. (B) Effects of nutrient starvation on Rubicon, p62 and LC3 in Huh7 cells. Huh7 cells were nutrient-starved for 2 or 6 hours as indicated and lysed for Western-blot analysis. The replicon cells were used as the control for comparison. (C) Colocalization analysis of GFP-LC3 puncta and lysosomes. Stable Huh7 cells that expressed GFP-LC3 were nutrient-starved for 2 hours and stained with Lysotracker-red for lysosomes. The HCV replicon cells were also stained with Lysotracker-red for comparison. (D) Colocalization efficiency of GFP puncta with Lysotracker-red shown in (C). The results represent the average of >30 cells. (E) Effect of Rubicon knockdown on parental Huh7 cells and HCV replicon cells. Huh7 cells and HCV replicon cells were treated with the control siRNA or the Rubicon siRNA for two days. Cells were then lysed for Western-blot analysis. (F) Relative HCV RNA levels as measured by real-time RT-PCR. HCV replicon cells treated with either the control siRNA or the Rubicon siRNA for two days were lysed for quantification of HCV RNA by real-time RT-PCR. (G) Effect of UVRAG overexpression on HCV replicon cells. HCV replicon cells were transfected with the control vector or flag-UVRAG plasmid for two days. Cells were then lysed for Western-blot analysis. (H) Relative HCV RNA levels as measured by real-time RT-PCR. HCV replicon cells transfected with either the control vector or the flag-UVRAG plasmid for two days were lysed for quantification of HCV RNA by real-time RT-PCR. In (F) and (H), *, p<0.05.
Departure of Armi from Yb bodies requires piRNA precursor loading onto Piwi.Figure 4Upper: Northern blotting shows that Flag-Piwi WT (WT) and PAZ mutant (PAZmt) but not MID mutant (MIDmt) bound with flam-piRNA precursors in Zuc-depleted OSCs. Endogenous Piwi was depleted simultaneously with Zuc (Zuc KD + Piwi KD). Zuc was depleted by RNAi and so residual Zuc may have remained in cells. Therefore, a tiny amount of piRNAs was loaded onto Piwi. F-Piwi WT, PAZmt, and MIDmt were made to be RNAi-resistant by mutagenesis (see Materials and Methods). Lower: The abundance of immunopurified Flag-Piwi (F-Piwi) is shown by western blotting. Armi localization to Yb bodies observed in Zuc- and Piwi-depleted OSCs (Zuc KD + Piwi KD) (Figs 3A and B) was restored by ectopic expression of Flag-Piwi WT and PAZmt but not of MIDmt (see also Fig EV4B). Armi, mitochondria, and F-Piwi are shown in green, red, and yellow, respectively. The scale bar represents 5 μm. DAPI (blue) shows the nuclei. Western blotting shows that Flag-Piwi (F-Piwi) WT (WT) and PAZ mutant (PAZmt) but not MID mutant (MIDmt) bound with Armi in Zuc/Piwi-depleted OSCs (Zuc KD + Piwi KD).
Study design.Figure 1Schematic of ctDNA detection in matched CSF, plasma and urine samples from HGG and LGG patients using INVAR and/or sWGS. HGG - high grade glioma, LGG - Low grade glioma, CNS - central nervous system, CSF - cerebrospinal fluid, sWGS - shallow whole genome sequencing.
Figure 1A, B Hypocotyl length of ChWT, (A) RNAi-ChHFR1 transgenic and (B) chfr1 mutant seedlings grown under different R:FR. Seedlings were grown for 7 days in continuous W (R:FR>1.5) or for 3 days in W then transferred to W supplemented with increasing amounts of FR (W+FR) for 4 more days, producing various R:FR. Aspect of representative 7-day old ChWT, RNAi-HFR1 and chfr1-1 seedlings grown in W or W+FR (R:FR, 0.02), as indicated, is shown in lower panel. Data information: Values are the means ± SE of three independent biological replicates relative to ChWT value at 0 h. Asterisks mark significant differences (Student t-test: ** p-value <0.01; * p-value <0.05) relative to ChWT value at the same time point. C, D Effect of W+FR exposure on the expression of PIL1, YUC8 and XTR7 genes in seedlings of ChWT, (C) RNAi-HFR1 and (D) chfr1 mutant lines. Expression was analyzed in 7-day old W-grown seedlings transferred to W+FR (R:FR, 0.02) for 0, 1, 4, 8 and 12 h. Transcript abundance is normalized to EF1α levels. Data information: Values are the means ± SE of three independent biological replicates relative to ChWT value at 0 h. Asterisks mark significant differences (Student t-test: ** p-value <0.01; * p-value <0.05) relative to ChWT value at the same time point.
Nucleosome abundance is low throughout the telomere-proximal chromosomal region.Figure 4A ChIP-seq reads mapped to TEL2R (shown in reverse orientation for consistency) B In silico analyses of TEL2R DNA sequence. Black line in top panel shows A/T content. Dotted line represents average A/T content in S. pombe (www.pombase.org) Bottom panel shows the percentage of poly(dA:dT) tracts (defined as a sequence of 5 or more nucleotides consisting only of A or T). Red and grey shaded areas show TAS regions and subtelomeric genes, respectively C Nucleosome prediction for TEL2R using prediction algorithm [33]
Figure 3(A) Venn diagram showing overlap between virophagy-related genes in HeLa cells (Orvedahl et al., 2011) and ZIKV-infected hNSCs (Tang et al., 2016). (B) RT-qPCR analysis of FA pathway genes at 48 h after ZIKV infection of hNSCs. Mean ± SEM of biological triplicates. *p < 0.05, **p < 0.01 by Student's t test. (C-D) RT-qPCR analysis of ZIKV mRNA 48 h after ZIKV infection of HeLa cells overexpressing FANCC (C) or FANCC-specific siRNA (D). Mean ± SEM of biological triplicates. **p < 0.01 by Student's t test. (E) Flow cytometry analysis of ZIKVE expression in mock-infected (red), ZIKV-infected (blue), ZIKV-infected siFANCC-transfected (orange), and ZIKV-infected siE2F4-transfected (green) hNSCs at 48 h post-infection. (F) ZIKV NS1 and FANCC immunostaining 48 h after ZIKV infection of hNSCs. Scale bar = 100 µm. (G) ZIKV NS1 expression 48 h after ZIKV infection of hNSCs expressing control siRNA (NTC) or siFANCC-targeting siRNA. Scale bar = 100 µm. (H-I) ZIKV NS1 expression 48 h after ZIKV infection of hNSCs expressing control (NTC) or siE2F4-targeting siRNA. Mean ± SEM of six imaging fields of biological triplicates, **p < 0.01 by Student's t test, Scale bar = 100 µm, (J) Immunoblot of macroautophagy (p62, LC3I/LC3II) and selective autophagy (FANCC, TOMM20) proteins 24 h after overexpression of FLAG-tagged ZIKV NS1, NS2B, NS3, NS4A, NS4B and NS5 in 293FT cells. (K) Analysis of E2F4 ChIP-seq datasets (Encode) showing direct chromatin-binding interaction between E2F4 and FA pathway genes in HeLa (GEO: GSM935365, Farnham lab), K562 (GEO: GSM935600, Farnham lab), and MCF10A (GEO: GSM: 935400, Struhl lab) cell lines. H3K9ac and H3K4me3 datasets identify promoter regions. The genomic region is displayed in log scale with the scale range identified in each panel (0-30). The summed interaction (Sum) across all three cell types is represented as a heatmap. (L-M) Heatmaps of RT-qPCR analysis of FA pathway genes (K) or essential selective autophagy genes (L) in hNSCs 48 h after transfection with control (NTC) or E2F4-targeting siRNAs or after infection with ZIKV MR766 and Paraiba strains. Color patterns in heat map indicate as red showing the highest expression and blue showing the lowest expression of genes.
Figure 3A. SNPs/kb in physical windows of increasing size (kb) around crossover midpoints (red), or for matched randomly chosen positions (grey). Printed above the plot for each window are circles coloured green if crossover SNPs/kb values are significantly different to random (P<0.05), or red if not (P>0.05) (Bonferroni adjusted t-tests). The population (Col×Ws, Col×Bur (Lawrence et al, 2019), Col×Ct, Col×Ler (Serra et al, 2018b; Rowan et al, 2019) and Col×CLC) is printed above each plot, and the number of positions analysed is printed inset. B. As for A, but analyzing the indicated Col/Ler transition and transversion polymorphisms around Col×Ler crossovers (Rowan et al, 2019) (red), or the same number of random positions (grey). C. As for A, but analysing Col/Ler SNPs/kb around crossovers from wild type, HEI10-OE, recq4a recq4b or HEI10-OE recq4a recq4b (Serra et al, 2018b). All populations were generated from Col×Ler hybrids. Printed beneath are the estimated average numbers of Class I and Class II crossovers per meiosis for each genotype, measured via genotyping-by-sequencing (Serra et al, 2018b; Ziolkowski et al, 2017). To the right are plots showing the significance of SNPs/kb differences between crossover sets, across the windows tested (Bonferroni adjusted t-tests).
Figure 3A Assessment of K63-type ubiquitination of FOXP3 upon expression of wild type and catalytically deficient TRAF6 mutants. 293T cells were transfected with the given combinations of vectors encoding wild type TRAF6, the enzymatically deficient L74H or C70A TRAF6 mutants, HA-FOXP3, and FLAG-labeled ubiquitin molecules possessing a single lysine residue (K63) that restrain the possible ubiquitin monomer linkages on polyubiquitinated proteins to the K63-type only. Cells were lysed and proteins modified by K63-ubiquitin chains were recovered from lysates by pulling down using a bead-immobilized K63-specific TUBE reagent. The presence of FOXP3 among these modified proteins was determined by immunoblotting as were levels of FOXP3 and TRAF6 in the pre-IP whole cell lysate (WCL). B Co-immunoprecipitation (co-IP) of FOXP3 with TRAF6 and its facilitation of K63-linked ubiquitination. 293T cells expressing the indicated constructs encoding HA-FOXP3 and FLAG-TRAF6 were lysed and incubated with bead immobilized anti-FLAG antibodies. FOXP3 molecules co-IPed in this manner were resolved by SDS PAGE and detected by immunoblotting with anti-HA antibodies. C　293T cells were transfected with the indicated combinations of expression constructs encoding HA-FOXP3, Myc-TRAF6, and FLAG-tagged ubiquitin molecules - either wild type (FLAG-Ub), or a variant with a K-to-R mutation at either lysine residue 48 (K48R) or 63 (K63R) responsible for preventing K48- and K63-type polyubiquitination, respectively. . D Endogenous, reciprocal co-IP of FOXP3 and TRAF6 in murine iTreg lysate. Naïve CD62Lhigh/CD25-/CD4+ T cells were obtained by FACS and activated with anti-CD3/CD28 antibodies (1ug and 2ug/ml, respectively) in the presence of IL-2 and TGFβ (100U/ml and 5ng/ml, respectively) for four days before lysis. Antibodies against TRAF6 (left) or FOXP3 (right) were used to pull down target proteins and their interaction partners, which were resolved under denaturing conditions and visualized by immunoblotting. E Degree of K63 ubiquitination in the cellular FOXP3 pools of Traf6fl/flFoxp3Cre+ and Traf6wt/wtFoxp3Cre+mice. CD4+/YFP+ cells from Traf6fl/flFoxp3Cre+ ("Traf6-/-") or wild type Traf6wt/wtFoxp3Cre+mice ("Traf6+/+") were isolated by FACS, lysed and either total FOXP3 (right) or K63-ubiquitin-modified proteins (left) were immunoprecipitated. Levels of polyubiquitinated FOXP3 were observed in each by probing with antibodies specific for anti-K63-ubiquitin and FOXP3, respectively, and levels of total FOXP3 and actin were also measured by immunoblot analysis of whole cell lysate.
DAPK phosphorylates beclin 1 on Thr 119 located within its BH3 domain.Figure 4(A) A bacterially purified catalytic domain of DAPK was incubated for 15 min at 30°C with increasing concentrations (5-10 nmol) of a peptide corresponding to the BH3 domain of beclin 1 (aa 108-127) and with the same peptide in which Thr 119 was substituted by alanine. An in vitro kinase assay was performed, and the reactions were applied to Whatman filters. Total levels of TCA‐insoluble counts were measured and plotted against substrate concentration. Data are the mean±s.d. of three experiments. (B) A model of the interaction between the BH3 domain of beclin 1, and the hydrophobic pocket of Bcl‐XL. The cyan arrow points to the groove in which phosphorylated Ser 113 can be accommodated. The black arrow points to the site that might be disrupted by phosphorylation of Thr 119. The magenta arrow points to the positively charged groove in which phosphorylated Ser 127 can be accommodated. (C) Flag‐tagged DAPK (60 ng) was incubated with GST-WT beclin 1 or with GST-T119A beclin 1 (1000 ng) in the presence of Ca2+, calmodulin and ATP for 30 min. GST‐beclin 1 levels were visualized by Ponceau S staining, and phosphorylation on Thr 119 was detected by a phosphoThr 119 antibody (Western blot). (D) HEK293 cells were co‐transfected with Flag‐tagged beclin 1 with or without ΔCaM DAPK. After 24 h, beclin 1 was immunoprecipitated from cells, using Flag antibodies, and the immunoprecipitates were reacted with phosphoThr 119 antibodies. The Ponceau staining shows equal amounts of immunoprecipitated beclin 1. The cell extract blots were reacted with haemagglutinin antibodies or with beclin 1 antibodies. CaM, calmodulin; DAPK, death‐associated protein kinase; GST, glutathione S‐transferase; HEK, human embryonic kidney; TCA, trichloro‐acetic acid.
Figure 5A. Phosphorylation kinetics of T532, T539 and S558 on Apc1-loop500 in anaphase extracts. MBP-fused Apc1-loop500 WT or its derivatives (∆11, deletion of 11 residues including the B56-binding motif) was incubated with Xenopus egg extracts in the presence of non-degradable cyclin B at 23˚C. The samples taken as indicated were analysed by SDS-PAGE and immunoblotting using phospho-specific antibodies (pT532, pT539 and pS558) or MBP antibody. To monitor CDK activity, Apc3 antibody was used (bottom). The signals of a phospho-specific antibody and MBP antibody on the same membrane were detected in the 800- and 700-nm channels, respectively. The two channels were pseudo-coloured (green; pT532, pT539 and pS558, red; MBP) and overlaid in the bottom panel (Overlay). The signal of pS558 was not detected in ∆11 mutant because ∆11 mutation includes S558. B. Quantification of (A). The intensities of WT at 50 min were arbitrarily set to 1.0. Error bars, SEM from three independent experiments. C. Phosphorylation kinetics of T532, T539 and S558 on Apc1-loop500 of the endogenous APC/C in Xenopus egg extract. Endogenous APC/C was immunoprecipitated with Apc3 MAb (AF3.1) beads at the indicated time points after mitotic induction by adding non-degradable cyclin B, analysed by SDS-PAGE and immunoblotting with phospho-specific antibodies, pT532, pT539 and pS558. Quantification of Fig EV5A. The intensities at 45 min were arbitrarily set to 1.0. Error bars, SEM from three independent experiments. D. Dissociation of Plx1-PBD from Apc1-loop500. MBP-fused Apc1-loop500 WT or its derivatives (E562A) was incubated in anaphase extracts supplemented with non-degradable cyclin B at 23˚C for 1 h, and further incubated with 10 µg of WT Plx1-PBD for 15 min. The complexes were isolated by amylose beads and incubated in the presence (78 nM) or absence of purified PP2A- B56γ at 23˚C for 30 or 60 min. The bound proteins were recovered by amylose beads, separated by SDS-PAGE and detected by Coomassie brilliant blue (CBB) staining. The intensities at 0 min were arbitrarily set to 1.0.
Muscle metabolomes of MIRAS, PEO and MELAS patients A - B Metabolomes of muscle of MIRAS (A) and PEO (B) patients; PLS-DA plots; VIP score plots of top 15 metabolites; volcano plots of all metabolites.Figure 4Metabolomes of muscle of MIRAS (A) patients; PLS-DA plots; VIP score plots of top 15 metabolites; volcano plots of all metabolites. Data information: a Significantly changed metabolites outside the FDR cut-off. b Metabolites not significantly changed between patients and controls. AMP, adenosine monophosphate NAD, nicotinamide adenine dinucleotide TCA, taurocholic acid Metabolomes of muscle of PEO (B) patients; PLS-DA plots; VIP score plots of top 15 metabolites; volcano plots of all metabolites. Data information: a Significantly changed metabolites outside the FDR cut-off. b Metabolites not significantly changed between patients and controls. car., carnitine NAD, nicotinamide adenine dinucleotide C Methyl cycle, transsulfuration and glutathione biosynthesis pathways changed in IOSCA, MIRAS, PEO and MELAS/MIDD patients. Circled text: metabolites changed in blood; red, increased; blue, decreased. Coloured text: metabolites changed in muscle; red, increased; blue, decreased. Selected for MELAS muscle were metabolites with the highest fold-change. D Relative values of metabolites in muscle of MIRAS, PEO and MELAS/MIDD patients compared to controls. All data represent mean ± SD. *P = 0.031, **P = 0.008 (two sample T-test). UDP, uridine diphosphate.
Figure 2Scatterplot of DamID-seq signals (log2 ratio) for FlyBase genes (left) or genome integrated TEs (right) in EGFP-KD (control, x-axis) and Piwi-KD (y-axis) samples. Both x- and the y-axis is a log2 ratio. Gypsy signal with the greatest decrease upon Piwi-KD is indicated and shown in genome browser view in (B). DamID-seq signal densities are shown for OSCs with EGFP- and Piwi-KD at genomic regions harboring gypsy insertions with the greatest decrease upon Piwi-KD (indicated in (A)). Neighboring regions are annotated with protein-coding genes and LTR TEs. Note that this distribution of the reads includes multi-mapped reads. Boxplots showing fold changes in the expression of piRNA target or non-target TEs based on RNA-seq upon Piwi-KD (left), and the frequency of piRNAs, which target piRNA target TEs or non-piRNA target TEs (right). Definition of piRNA target and non-target TEs is in Fig EV1E. Boxplot whiskers, box, and central band show maximum, third quartile, median, first quartile, and minimum, respectively (n=10 for piRNA target TEs, n= 80 for non-piRNA target TEs). P-values were calculated using the Wilcoxon rank-sum test. Scatterplot as in (A) for EGFP-KD (control, x-axis) or Piwi-KD (y-axis) samples. Values were calculated for 90 TE consensus sequences (one value per one type of TE) (left). Scatterplot of RPM values for the same 90 TE consensus sequences in EGFP-KD (control, x-axis) or Piwi-KD (y-axis) samples examined by RNA-seq (right). The signals for mdg1, gypsy, and roo are labelled within the plots. Boxplot showing DamID-seq signals of piRNA target or non-target TEs upon EGFP (control)- or Piwi-KD. Boxplot whiskers, box, and central band show maximum, third quartile, median, first quartile, and minimum, respectively (n=10 for piRNA target TEs, n= 80 for non-piRNA target TEs). P-values were calculated using the Wilcoxon rank-sum test. Density plots for DamID-seq signals over consensus sequences from piRNA target (gypsy and mdg1, orange) and non-target TEs (roo, blue) in EGFP (control)- or Piwi-KD cells. Dot plot showing the correlation between the normalized density of indicated TEs per LAD (x-axis) and changes in Lam-Dam / Dam signals upon Piwi-KD (y-axis). Trend lines are in red. Spearman's rank correlation rho is indicated along with the p-value (algorithm AS 89). Oligo-FISH images for the representative piRNA target region described in Fig 4A, upon EGFP (control)- or Piwi-KD. Gray indicates DAPI staining for DNA, magenta indicates FISH signal, and green indicates Lamin immunofluorescence staining. Scale bar, 5μm. Ratio of distance from FISH signal to the nuclear periphery (DAPI surface) is quantified at right, dot in the box plot indicates mean value; Boxplot whiskers, box, and central band show minimum, first quartile, median, third quartile, and maximum, respectively. P-values calculated with Mann-Whitney U test. Diagram illustrating relocation of piRNA-target TE coding chromosome regions away from the nuclear periphery.