EMBO/sd-character-level-ner huggingface dataset:
Figure 7A-D Control: Double immunolabelling of SYCP3 (cyan) and α-Tubulin (red) on control squashed spermatocytes for the first and the second meiotic divisions at metaphase I (A) and metaphase II (B). Double immunolabelling of PCNT (green) and α-Tubulin (red) on control squashed spermatocytes at metaphase I (C) and metaphase II (D). Chromatin has been stained with DAPI (pseudo coloured in grey). E-H Plk1 (∆/∆) mouse model: Double immunolabelling of SYCP3 (cyan) and α-Tubulin (red) in monopolar spindle I (E) and monopolar spindle II (F). Double immunolabelling of PCNT (green) and α-Tubulin (red) labelling in monopolar spindle I (G) and monopolar spindle II (H). Chromatin has been stained with DAPI (pseudo coloured in grey). I-L BI2536-treated spermatocytes: Double immunolabelling of SYCP3 (cyan) and α-Tubulin (red) at monopolar spindle I (I) and monopolar spindle II (J). Double immunolabelling of PCNT (green) and α-Tubulin (red) labelling in monopolar spindle I (K) and monopolar spindle II (L). Chromatin has been stained with DAPI (pseudo coloured in grey). Cells shown were treated for 100µM BI2536 for 8h treatment on organotypic cultures of seminiferous tubules. M-P MLN8237-treated spermatocytes: Double immunolabelling of PCNT (green) and α-Tubulin (red) in monopolar spindle I (M) and monopolar spindle II (N). Double immunolabelling of PCNT (green) and α-Tubulin (red) in monopolar spindle I (O) and monopolar spindle II (P). Chromatin has been stained with DAPI (pseudo coloured in grey). Cells shown were treated for 10µM MLN8237 for 8h treatment on organotypic cultures of seminiferous tubules. Q-R Quantitative analysis of the incidence of monopolar spindles during the first (Q) and second (R) meiotic divisions for each experimental condition. Percentage of monopolar spindles functional analysis are represented for 100µM BI2536 and 10µM MLN8237 for 8h treatments on organotypic cultures of seminiferous tubules. Experiments were conducted in three different biological replicates.
The B-TFIID cofactor BTAF1 is a direct miR-132 target in CD4+ T cells.Figure 2A. Volcano plot (Log2(Fold Change) vs -Log(P value)) of RNA gene expression in purified naïve CD62L+ CD44- WT and miR-132-/- CD4+ T cells. Fold change determined as log2 mean FPKM (miR-132-/-/WT) from 4 WT and 4 miR-132-/- mice. Transcripts significantly different between WT and miR-132-/- cells (p<0.05) shown in red. B. Volcano plot of RNA gene expression in purified naïve CD62L+ CD44- WT and miR-132-/- CD4+ T cells following 18hr in vitro stimulation with anti-CD3/anti-CD28 under Th1 conditions. Fold change determined as log2 mean FPKM (miR-132-/-/WT) from 4 WT and 4 miR-132-/- mice. Transcripts significantly different between WT and miR-132-/- cells (p<0.05) shown in red. C. Volcano plot of transcripts containing a conserved miR-212/132-3p target site in naïve CD4+ T cells from WT or miR-132-/- mice. D. Volcano plot of transcripts containing a conserved miR-212/132-3p target site in in vitro polarised (Th1 condtions, 18h post stimulation) CD4+ T cells from WT or miR-132-/- mice. E. Volcano plot of transcripts containing a conserved miR-212/132-3p target site in spleen CD4+ T cells from d28 L. donovani infected WT or miR-132-/- mice. F. BTAF1 transcript levels determined by qRTPCR in WT (blue) or miR-132-/- (red) in naïve (d0) and Th1 polarised for 18h (d1) CD4+ T cells, and CD4+ T cells from d28 L. donovani infected WT or miR-132-/- mice. N=8-9 for each WT and miR-132-/ Data information: Significance in (F) determined by unpaired t-test. * p<0.05, ** p<0.01. G. Expression of BTAF1 protein in d0 naïve and d1 (18hr) Th1-polarised WT and miR-132-/- CD4+ T cells, as determined by Western blot. Each lane from individual mouse, and representative of two independent experiments. H. Relative luciferase activity in HeLa transfected with plasmid containing WT BTAF1 3'UTR (white) or BTAF1 3'UTR on which the miR-132 binding site is mutated (grey) downstream of renilla luciferase, in the presence of miR-132-3p or miR-212-3p mimics. Error bars indicate SEM from eight replicate treatments. Data information: Significance in (H) determined by unpaired t-test. * p<0.05, ** p<0.01.
Figure 1A. Confocal images of live control (top) and U18-treated (bottom) tsA201 cells expressing P4M-YFP. Black and orange rectangles show expanded views of PtdIns4P in perinuclear and endo/lysosomal compartments, respectively. B. Quantification of perinuclear P4M-YFP mean fluorescence intensity normalized to the cytoplasm. Control: n=19; U18: n=16. C. Confocal images of control and U18-treated hippocampal neurons transfected with P4M-YFP. The inset panels show expanded views of PtdIns4P in perinuclear regions. D. Quantification of perinuclear YFP-P4M mean fluorescence intensity normalized to the cytoplasm. Control: n=11; U18: n=10. E. Control (top) and NPC1I1061T (bottom) patient fibroblasts transfected with P4M-YFP (left), TGN38-RFP, and merge (right). Black rectangles show expanded views of PtdIns4P in TGN38 regions. Dashed white lines were used for intensity profile analysis (F). F. Line scan analysis taken from white dashed lines in (E) showing intensity changes of P4M and TGN38 signal from control (top) and NPC1I1061T (bottom) cells. G. Quantification of P4M signal in TGN38 region (P4MTGN38). Control: n=12; NPC1I1061T: n=19. H Same as (E) only LAMP1-CFP expressed instead of TGN38. I. Same as (F) only LAMP1 instead of TGN38. J. Quantification of P4M signal in LAMP1 regions (P4MLAMP1). Control: n=14; NPC1I1061T: n=16. K. Heat map of PtdIns, PtdInsP and PtdInsP2 levels from 4 different murine brain regions. The colors from green to red indicate the relative contents of lipid isoforms in different brain regions from NPC1I1061T mice relative to wild-type brains; n=3 for both WT and NPC1I1061T animals.
Lamps levels in livers from PPCA(−/−) mice.Figure 4(A) Immunoblot analysis for lamp2a, all lamp2s, lamp1, transferrin receptor (TFR) or cathepsin D (cath D) of liver homogenates (100 μg of protein) from wild‐type (WT), PPCA(−/−) and β‐galactosidase(−/−) (GM) mice. Right: densitometric quantification of three immunoblots (means + SE) similar to the ones shown here. Differences from the control value were significant to P 0.001 (**) and P 0.05 (*). (B) Immunocytochemistry for lamp2a (left), lamp1 (center) or lamp2s (right) in hematoxylin and eosin‐stained sections from the same livers. Bar: 50 μm. Black arrows: hepatocytes; white arrows: Kupffer cells. (C) Immunofluorescence for lamp2a (top) and P‐glycoprotein (bottom) in mouse liver sections. The right panel shows merged images. Bar: 100 μm. Inset shows detailed area at higher magnification. Bar: 50 μm.
Figure 2(A~C) HeLa cells stably expressing GFP-DFCP1 were transfected with or without PAQR3-specific shRNA. GFP signals were observed in these cells before or after glucose starvation (GS, shown in A). The knockdown efficiency of PAQR3 is shown in B and the statistics of GFP-positive puncta per cell is illustrated in C. Values are presented as mean ± SD, *** P < 0.001. Scale bar: 10 μm. (D) GFP-WIPI1 was transfected into WT or PAQR3-deficient MEFs. 24 h later, the cells were incubated under GS for 4 h, followed by fluorescence observation. Scale bar: 10 μm. (E) Different VPS34 complexes of WT or PAQR3 knockout HeLa cells were immunoprecipitated by ATG14L and UVRAG antibodies respectively in normal medium (NM) or GS. Then PI(3)P level was determined by a quantitative ELISA assay. The PI(3)P level was normalized to the amount of ATG14L or UVRAG used in the assay (n = 5; ***p < 0.001; ns, not significant). (F) HeLa cells infected with control or PAQR3-overexpressed lentivirus were treated with GS for 4 h. PI(3)P levels were determined as in Figure 2E(n = 5; **p < 0.01; ns, not significant). (G, H) PAQR3 knockout HeLa cells were transfected with plasmids of multiple PAQR3 deletion mutants. At 24 h after the transfection, the cell lysates were used to immunoprecipitate VPS34 complex by ATG14L antibody, followed by immunoblotting. The activity of ATG14L-linked VPS34 activity was detected by a quantitative ELISA assay. The PI(3)P level was normalized to the amount of ATG14L (n = 5; **p < 0.01).
SIRT7 modulates levels of H3K18Ac at DSB.Figure 6A, B H3K18Ac IF in WT and SirT7-/- fibroblasts. Cells were pulsed with EdU (green), stained with antibody against H3K18Ac (magenta) and then counterstained with DAPI (blue). (A) Representative IF images of late S-phase nuclei (EdU positive; scale bar 5µm). (B) Quantitation of (A) throughout the cell cycle. C Same as in (B), in S-phase cells at the indicated times after IR (1Gy) (mean ± SEM of >30 cells per cell cycle, time point, and genotype). D Laser-induced DNA damage in HT1080 cells expressing HA-tagged SirT7 stained with antibodies against γH2AX (red) and HA (green), then counterstained with DAPI (blue) 30 min post damage (scale bar 3 µm). E ChIP assays of SIRT7 enrichment at the indicated loci as described in Figure 5H (mean ± SEM; one of two independent experiments is shown). F Recruitment kinetics of GFP-tagged SirT7 after laser-induced microirradiation. (Top) Representative cell at the indicated times after induction of DNA damage (white circle; scale bar 2µm). Images were adjusted to account for photobleaching by normalizing to nucleoplasmic background signal. (Middle) Quantitation of recruitment kinetics at the site of induced damage in the presence of 5M KU-55933 ATM inhibitor (ATMi), 10M Olaparib PARP inhibitor (PARPi), or DMSO. KU-55933 and Olaparib were added 30 min and 1 hour respectively prior to DNA damage (mean ± SEM; sample size: SIRT7-GFP, n=34; SIRT7-GFP + ATMi, n=36; SIRT7-GFP + PARPi=18). (Bottom) Same as (middle) except quantitation of SIRT7-GFP relative intensity within the nucleolus. Data was acquired at five second intervals over a span of five min. G ChIP assays of H3K18Ac enrichment at the indicated loci as described in Figure 5H. H3K18Ac enrichment adjacent to the induced DSB in control and SIRT7 depleted cells (bottom left), or in cells overexpressing SIRT7 in the presence or absence of PARP inhibitors (PARPi) (bottom right) (mean ± SEM; one of two independent experiments is shown).*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 by ANOVA Single Factor.
Figure 4A. Recombinant PP1 was inhibited by H2O2 (0.2 mM) and activity was not restored by glutathione (GSH), cysteine (Cys) or dithiothreitol (DTT). A Cys127Ser/Cys273Ser PP1 mutant was inhibited by H2O2 similarly to wild-type PP1. Values above bars denote level of significance for the inhibitory effect of H2O2. B. Ascorbate (Asc) dose-dependently restored PP1 activity. #, significant effect of Asc compared to H2O2 alone. C. EPR spectra of PP1 incubated with ascorbate (1 mM) alone (a) or PP1 exposed to H2O2 followed by catalase treatment, then incubation with ascorbate (b). Panel b shows a typical spectrum for the ascorbyl radical (hyperfine splitting constant, aH = 1.8G), similar to the positive control obtained by exposing ascorbate to H2O2 (c). Panel d shows that no ascorbyl radical is detected if H2O2 is degraded by catalase in the absence of PP1, prior to ascorbate addition. D. Cartoon representation of the active site of H2O2-treated PP1 as in Fig EV2D. 2mFo-DFc electron density map at the 2.2 Å resolution is shown in yellow at the 1.1 level. H2O2-treatment causes an overall shrinkage of the PP1 coordination sphere by 0.12 Å compared to ascorbate-treated crystals consistent with the oxidation of the dinuclear center. This increases the energy barrier for the catalytic steps involving -OH- attack on the phosphorous center of the bridging phosphate and rupture of the P-O scissile bond with the assistance of H125 (black arrows). Reported coordination distances in Å are averaged over the two PP1 molecules in the crystallographic asymmetric unit. See also Appendix Table S1. E. Effect of Nox4 on GADD34/PP1-mediated eIF2 dephosphorylation in transfected HEK293 cells. Nox4 increased eIF2 phosphorylation in cells transfected with GADD34 and WT PP1, and resulted in even higher phospho-eIF2α levels in cells transfected with N124D or D64N PP1 variants. F. Effect of ascorbate (Asc, 0.5 mM) on phosphatase inhibition in tunicamycin-treated H9c2 cells with overexpression or knockdown of Nox4 (Ad.Nox4 and Ad.shNox4, respectively). In control cells, ascorbate enhanced tunicamycin-stimulated increases in phosphatase activity. Phosphatase activity was lower in Nox4-overexpressing than control cells but was normalized by ascorbate to the same level as in control cells. In Nox4 knockdown cells, tunicamycin-induced increases in phosphatase activity were enhanced and ascorbate had minimal additional effect.
Figure 2(A) Representative images of hearts from LC or CMNKO mice subjected to sham operation or AAC for 2 weeks. Scale bar: 0.5cm. (B) VW/BW of LC or CMNKO mice 2 weeks after sham operation or AAC. n=9:9:16:15. (C) Representative H&E staining of cross sections of left ventricles. Scale bar: 25μm. (D) Representative WGA staining. Scale bar: 10μm. (E) Quantification of cardiomyocyte size. n=10:10:8:9. (F) qRT-PCR analysis of hypertrophy-related genes in left ventricles. n=8:7:15:15. (G) Representative picrosirius red staining of cross sections of left ventricles. Scale bar: 50μm. (H) Quantification of fibrotic areas. n=11:10:10:9. (I) qRT-PCR analysis of fibrosis-related genes in left ventricles. n=8:7:15:15. (J) Ejection fraction measured by echocardiography. n=9:7:14:12. (K) Fractional shortening measured by echocardiography. n=9:7:14:12. (L) Lung weight to body weight ratio (LW/BW) of LC or CMNKO mice 2 weeks after sham operation or AAC. n=8:7:16:15. (M) Cumulative survival rate of LC or CMNKO mice subjected to AAC. n=12:10. Two-way ANOVA followed by Bonferoni post-tests was used for statistical analysis.
Figure 4A) Schematic illustration of iAID-conditioned Cdc55 depletion system: Cdc55 is fused with an AID (Auxin-inducible degron) tag rendering the protein sensitive to auxin (Morawska and Ulrich, 2013; Nishimura et al., 2009). Expression of AID-Cdc55 is controlled by a TetOff promoter and is repressed in the presence of doxycycline. Upon addition of auxin Cdc55 is rapidly depleted. B) Western-blot illustrating the depletion of AID-Cdc55 at various time points after addition of auxin (upper panel); Pgk2 serves as a negative control (lower panel). (C) Scatter plot of log2 fold-changes of overlapping phosphorylation sites from setup cdc55Δ (x-axis) and the Cdc55-depletion system (y-axis). Stress-independent sites are shown in grey, stress-induced sites are shown in black. The dotted lines mark two-fold thresholds. (D) Transcriptional analysis of stress-induced expression of ESR genes CTT1 (upper panel) and PGM2 (lower panel) under different strain conditions (i.e. presence/absence of IGO1/2 and induction of iAID-Cdc55 depletion). Measured time points after stress application are depicted on the x-axis, target gene mRNA levels are illustrated on the y-axis as fold over reference gene IPP1, values are normalized relative to wild type without induction of iAID-Cdc55 depletion at 30 minutes post-stress. E) Transcriptional analysis of stress-induced expression of ESR genes CTT1 (upper panel) and PGM2 (lower panel) at 30 minutes post-stress. Target gene mRNA levels are illustrated on the y-axis as fold over reference gene IPP1, values are normalized relative to wild type without induction of iAID-Cdc55 depletion. dep.: Cdc55 depleted, n.d.: Cdc55 non-depleted.
Figure 4In wild-type ESCs, ID1 binds to ZRF1 and to other interactors (indicated as X). In self-renewing conditions, ID1 impairs ZRF1 binding to chromatin. Upon differentiation, Id1 expression decreases and ZRF1 is recruited to neural genes promoting their transcriptional activation by displacing Polycomb repressive complexes (PRC). Id1 over-expression blocks ZRF1 binding to chromatin, thus impairing the expression of neural genes during differentiation. In Id1KO ESCs, ZRF1 is bound on Polycomb target genes involved in neural differentiation, and promotes their expression. Correct expression of neural genes in Id1KO ESC is restored upon depletion of Zrf1.
Figure 3(A) Representative image of the intestinal populations purified in the cell sorting experiments. (B) IF analysis of EphB2 in P6 WT and IκBα KO intestine. Data information: Scale bars , 25 μm. (C) P-IκBα IF of EphB2-high or negative sorted cells. Data information: Scale bars , 25 μm. (D) Clustering analysis of the indicated cell populations based on their transcriptional profiles obtained in RNA-seq. 3 mice per genotype were initially processed although 1 EphB2neg KO replicate was excluded from the analysis due to insufficient number of reads in the RNA seq. (E) Correlation plot of the indicated differentially expressed gene (DEG) sets. Values for DEG in fetal and adult ISC was obtained from Nusse et al, 2012. (F) Table indicating the top 15 up-regulated genes in the IκBα KO EphB2-high signature including 11 genes in the fetal ISC signature (in orange). (G) GSEA of EphB2-high cells indicating the enrichment of relevant ISC-related pathways. (H) Representative image of the Lgr5+ population purified in the cell sorting experiments. (I) qPCR analysis of the indicated genes in purified Lgr5+ cells from the different IκBα backgrounds. 4 WT and 1 KO mice were analyzed with at least three technical replicates. Bars represent mean values ± standard error of the mean (s.e.m). p values were derived from an unpaired t-test, two-tailed, ****p-value<0.0001, **p-value<0.005, *p-value<0.05, n.s. no significant.
Replication of the legA9 mutant is not due to extended macrophage survival.Figure 8(A) BMDMs were not infected (NT) or infected with L. pneumophila JR32 or the legA9 mutant at an MOI of 0.5, 1 or 5 for 24 h. Apoptosis was quantified by Photometric enzyme immunoassay analysis of cytoplasmic (apoptosis) histone−associated−DNA fragments (ELISA). (B) BMDMs were either not treated (NT) or treated with L. pneumophila JR32 or the legA9 mutant at an MOI of 0.5 or 5 for 24 h. The fold change in LDH release was measured from the overall population of macrophages. (A-B) Data are shown as mean ± SD of n = 3 and are representative of three independent experiments.
Endogenous TMEM59 mediates LC3 activation and interacts with ATG16L1 in response to S.Figure 9(A) Depletion of TMEM59 inhibits LC3II generation by SA at early infection times. HeLa cells were transfected with the indicated siRNAs and, 48 h later, infected with the bacteria (SA) for the shown times before lysing them for western blotting against the indicated molecules. The right panel shows successful TMEM59 depletion (rabbit anti‐TMEM59 immunoprecipitation plus chicken anti‐TMEM59 WB). (A) The right panel shows successful TMEM59 depletion (rabbit anti‐TMEM59immunoprecipitation plus chicken anti‐TMEM59WB). (B) Colocalization events between bacteria (GFP labelled), TMEM59-HA and Cherry-LC3A in infected cells. HeLa cells stably expressing TMEM59-HA and Cherry-LC3A were infected for the indicated times (moi=10), and processed for anti‐HA immunofluorescence. Representative confocal images are shown. The GFP, anti‐HA (Cy5) and DAPI signals were pseudocolored in blue, green and white, respectively. Asterisks indicate overlay images: (*), Cherry-LC3A/TMEM59; (**), Cherry-LC3A/TMEM59/bacteria; (***), Cherry-LC3A/TMEM59/bacteria/DAPI. (C) Colocalization between bacteria, TMEM59-HA and Cherry-ATG16L1. HeLa cells stably expressing TMEM59-HA and Cherry-ATG16L1 were infected with SA (2 h, moi=10), and processed for anti‐HA immunofluorescence. Shown are confocal pictures displaying two representative examples. Asterisks indicate overlays as in B. (D) SA promotes co‐precipitation of endogenous ATG16L1 with endogenous TMEM59. HeLa cells were infected with SA (2 h, moi=10), lysed and subjected to immunoprecipitation with anti‐TMEM59 antibodies or irrelevant protein G beads (as indicated). Immunoprecipitates were processed for western blotting. The right panel shows control WBs of the lysates used for immunoprecipitation. (E) Depletion of TMEM59 reduces recovery of SA from infected cells. HeLa cells were transfected with the indicated siRNAs, infected with SA 96 h later (5 h, moi=0.1) and lysed for CFU evaluation. The experiment was carried out three independent times. The graph displays average CFU counts obtained for the 10−1 dilution of the relevant extracts, ±s.d. of the three data sets. Asterisks indicate significant differences (P<0.01, paired Student's t‐test). (E) Depletion of TMEM59 reduces recovery of SA from infected cells. HeLa cells were transfected with the indicated siRNAs, infected with SA 96 h later (5 h, moi=0.1) and lysed for CFU evaluation. The experiment was carried out three independent times. The right panel shows successful TMEM59 depletion (anti‐TMEM59 immunoprecipitation plus anti‐TMEM59 WB).Source data for this figure is available on the online supplementary information page.
Increased accumulation of insoluble protein aggregates in the atg7 and nbr1 mutants under heat stress.Figure 12(A) Accumulation of insoluble proteins. Leaf tissues from wild-type (WT), atg7 and nbr1 mutants collected at indicated hours (h) under 45°C for preparation of total, soluble and insoluble proteins as described in Materials and Methods. Total proteins in the starting homogenates and insoluble proteins in the last pellets were determined the percentages of insoluble proteins to total proteins were calculated. (B) Profiles of soluble and insoluble proteins. Proteins from the first supernatants (S) and last pellets (P) were subjected to SDS-PAGES and stained with Coomassie brilliant blue. Major proteins accumulated in the pellets from heat-stressed atg7 mutant plants were indicated by arrows.