EMBO/sd-character-level-ner huggingface dataset:
Figure 5Cryo-EM visualizes tRNA-bound nascent chain (red, NC) within the ribosomal tunnel (A) and a density between separated TMHs 2b and 7 of the lateral gate (B, shown in mesh). The proximate cysteine positions within SecY used for cross-linking are indicated and highlighted in red. Western-blot against the hemagglutinin tag within the nascent chain FtsQC40 reveals a cross-linking product of ~80 kDa in presence of nanodisc-reconstituted SecYC282EG and SecYC283EG, presumably assigned to FtsQxSecY adduct. The double band for FtsQC40 likely indicates the incomplete cleavage of the hexa-histidine tag by 3C protease. In-gel fluorescence imaging of SecYCF488A reveals a cross-linking product of ~80 kDa assigned to the FtsQC40xSecYC283 adduct ("SecYxFtsQ"). Bands for covalently cross-linked SecY dimer ("SecYxSecY"), and adducts of SecY and ribosomal proteins ("SecYxRiboP") are indicated. The nascent chain-specific adduct "SecYxFtsQ" does not depend on the presence of PE lipids. FtsQ:SecY cross-linking is position specific. FtsQ TMH C-terminal residues 40-42 could be cross-linked to the lateral gate (SecY residue 283), while no adduct could be detected at the upstream position 35. A refined scheme of the nascent chain insertion via the translocon. In the early intermediate state, the ribosome binding to the loop 6/7 and the emerging nascent chain cause displacements of SecY TMHs 2b and 7 at the cytoplasmic interface and "unzip" the lateral gate. The rigid-body tilt of TMH 7 leads to a close contact between TMHs 3 and 7 at the periplasmic side, so the V-shaped crevice is formed. The flexible nascent chain reaches the core of the lateral gate and gradually egresses the lipid moiety, where the helical fold is stabilized by hydrophobic interactions. Upon further nascent chain elongation and movements of SecY TMHs 2b, 3, 7 and 8, the widely open "inserting" state of the translocon is formed, with the complete nascent TMH at the lateral gate prior the release into the membrane.
mTORC1 activation promotes ROS-dependent DDR and contributes to the senescent phenotype via PGC1-β dependent mitochondrial biogenesis.Figure 5A) ROS levels (DHE intensity) and mean number (N) of yH2A.X foci after mTOR knockdown (72 hours) in proliferating and senescent (2 days after 20Gy X-ray) MRC5 fibroblasts. Data are mean±S.E.M of n=3 independent experiments; Asterisks denote statistical significant P<0.05 One-way ANOVA. B) ROS levels (DHE intensity) and mean number (N) of yH2A.X foci in proliferating and senescent (3 days after 20Gy X-ray) MRC5 fibroblasts treated with or without 100nM Rapamycin and/or 2.5mM of the antioxidant NAC. Data are mean±S.E.M of n=3 independent experiments; Asterisks denote statistical significant P<0.05 One-way ANOVA. C) (top) Scheme illustrating the experimental design: human MRC5 fibroblasts were irradiated with 20Gy X-ray and treated at day 3 after IR with 10µM of the ATM inhibitor (ATMi) KU55933 and/or 100nM Rapamycin (Rap); (bottom) Effect of single or combined inhibition of ATM and mTORC1 on the mean number (N) of γH2A.X foci, Sen-β-Gal activity and p21 expression in proliferating and senescent (10 days after 20Gy X-ray) MRC5fibroblasts. Data are mean±S.E.M of n=3 independent experiments. Western blots are representative of 3 independent experiments; Asterisks denote statistical significant P<0.05 One-way ANOVA. D) ROS levels (DHE intensity) in proliferating and senescent (3 days after 10Gy X-ray) wild-type and PGC-1β-/- MEFs (top) and PGC-1β overexpressing MEFs (bottom). Data are mean±S.E.M. of n=3 independent experiments; Asterisks denote statistical significant P<0.05 One-way ANOVA. E) (top) Representative images and quantification of immunofluorescence staining against FLAG-PGC-1β (red) and 53BP1 (green) in proliferating and senescent (2 days after 10Gy X-ray) PGC-1β overexpressing MEFs cultured at 3% O2, with or without 250µM of the antioxidant Trolox (scale bar=10µm). Data are mean±S.E.M, n=3 independent experiments; Asterisks denote statistical significant P<0.05 One-way ANOVA. F) (top) Effect of overexpression of mutated Rheb (N153T) and (bottom) effect of 100nM rapamycin on the mean number (N) of 53BP1 foci in proliferating and senescent (3 days after 10Gy X-ray) wild-type and PGC-1β -/- MEFs. Data are mean±S.E.M of n=3 independent experiments (at least 125 cells were analyzed per condition). Asterisks denote statistical significant P<0.05 One-way ANOVA.
a) Schematic representation of human PINK1 (MTS, mitochondrial targeting sequence; TM, transmembrane domain; Ser/Thr-Kinase, Serine/threonine kinase domain).figf3(b) Co-immunoprecipitations of Parkin and PINK1 after mitochondrial depolarization. Endogenous Parkin and PINK1 were co-immunoprecipitated from neuronal SH-SY5Y cells after CCCP treatment with specific antibodies. (c) Western blot of PINK1 siRNA-transfected HeLa cells used in d-h demonstrates the knockdown efficiency and specificity of PINK1 silencing. d-h) PINK1-specific siRNA abrogates Parkin translocation to mitochondria after 2 h, and mitochondrial clearance after 24 h, of CCCP treatment. This can be rescued by re-transfection with wild-type PINK1, but not with a kinase-dead mutant or a PINK1 variant lacking the MTS. Control siRNA- or PINK1 siRNA-silenced HeLa cells were transfected with EGFP-Parkin wild-type and PINK1-V5 wild-type, 3×KD or ΔMTS mutants. Use of appropriate empty vector controls is depicted. Cells were treated with CCCP and stained with anti-TOM20 (pseudo coloured; blue) and anti-V5 (red) to visualize mitochondrial morphology and re-transfected PINK1, respectively. Wild-type Parkin was visualized by EGFP epifluorescence (green). Parkin-positive cells are surrounded by a line. Representative images of four independent experiments are shown. Scale bars, 10 μm. (e) Wild-type Parkin colocalization with impaired mitochondria after 2 h of CCCP treatment was analysed (n = 4). (g) Quantification of results from cells in e. (f) Uncleared mitochondria were analysed after 24 h of CCCP treatment (n = 4). (h) Quantification of results from cells in f. Data represent the mean ± s.d., **P ≤ 0.005, ***P ≤ 0.0005 compared with control siRNA-transfected cells. Immunostaining of control siRNA-transfected cells at all analysed time points is shown in Supplementary Information, Fig. S2. WT, wild-type.
PlcA/B‐dependent accumulation of NDP52+ granules in Listeria‐infected cells.Figure 4(A) HeLa cells left unstimulated (CTR) or infected with Listeria WT for 1-4 h, or with Listeria TKO for 4 h and with analysed by IF using an antibody against NDP52. (B) Higher magnification view of the selected area from (A) showing that NDP52+ granules generally form at the vicinity of cytosolic Listeria. (C) Percentage of cells infected with Listeria WT for 1-4 h, or with Listeria TKO for 4 h, displaying one or several NDP52+ granules. Values are means±s.e.m. n=3. (D) Percentage of cells infected for 4 h with different Listeria mutants as indicated displaying one or several NDP52+ granules. Values are means±s.e.m. n=3. (E) HeLa cells infected with Listeria WT, PlcA/B− or PlcA/B− ectopically expressing PlcA for 3 h, analysed by IF using an antibody against NDP52. (F, G) Percentage of cells infected for 3 h with Listeria WT, PlcA/B− or PlcA/B− ectopically expressing PlcA displaying one or several NDP52+ granules (F) or bacteria entrapped in NDP52+ autophagosomes (G). Values are means±s.e.m. n=2. Scale bars: 5 μm.
Figure 5Left panel: flag-tagged full-length HDAC1 was overexpressed with myc-tagged TDP-43 in HEK-293T cells; the cell lysates were immunoprecipitated for flag and immunoblotted for TDP-43 and flag. Right panel: myc-tagged TDP-43 was overexpressed with flag-tagged full-length HDAC1 in HEK-293T cells; the cell lysates were immunoprecipitated for myc and immunoblotted for flag and TDP-43. Upper left: flag-tagged full-length HDAC1 (b.Ⅰ) or various truncation mutations (b.Ⅱ-Ⅳ) were overexpressed with myc-tagged TDP-43; the catalytic domain is indicated in red. Lower panel: the western blotting of cell lysates immunoprecipitated for flag and immunoblotted for TDP-43. Upper panel: Immunoprecipitation of cytosolic HDAC1 and immunoblotting of HDAC1 and TDP-43 in WT and FTLD-TDP Tg mice. Lower histogram: Quantification of immunoprecipitation results of HDAC1 and TDP-43 in WT and Tg mice. N = 5 mice per group, data are presented as mean ± SEM (%), * p =0.0149, *** p =0.0003 by t-test. Western-blot of HDAC1 and TDP-43 in urea-soluble fractions. N = 5 mice per group.
Figure 6 A-E G1-arrested cell cultures were released in YEPD supplemented with CPT (50 μM). (A, D) Electron micrographs of a representative reversed fork with the four-way junction magnified in the inset (A  A-E G1-arrested cell cultures were released in YEPD supplemented with CPT (50 μM) (B, C) Frequency of reversed replication forks in CPT (40 minutes, 50 μM CPT). Two independent experiments were performed with very similar results. The results of the individual biological replicates and the number of analyzed molecules are shown in Fig EV4C  A-E G1-arrested cell cultures were released in YEPD supplemented with CPT (50 μM). (A, D) Electron micrographs of a representative reversed fork wit a three-way replication fork exposing ssDNA at the junction (D). In (D) the black arrow indicates the ssDNA region, which is magnified in the inset. Scale bars: 200nm, 50nm in the insets  A-E G1-arrested cell cultures were released in YEPD supplemented with CPT (50 μM) (E) Graphical distribution of ssDNA length at the junction (black arrow in (D)) in forks isolated after CPT treatment (40 minutes, 50 μM CPT). Only molecules with detectable ssDNA stretches are included in the analysis. The lines show the median length of ssDNA regions at the fork in the specific set of analyzed molecules. Statistical analysis: Mann-Whitney test; ns, not significant; *, P< 0.05; **, P< 0.01; ****, P< 0.0001. The number of analyzed molecules is in brackets  F, G G1-arrested cell cultures (αf) were released into YEPD supplemented with CPT (50 μM). Western blot analysis with anti-Rad53 antibodies
Figure 5(A) Representative tracing path of WT mice and KO mice in the open field test. (B) The total traveling distance was not different between WT and KO mice. (C) KO mice spent less time in the center than WT mice. Date information: All mice were male, 8-12 weeks old. WT, n=12 mice; KO, n=12 mice. Error bars represent the means ± S.E.M.; Two-tailed unpaired t-test, P < 0.05 (*), P < 0.01(**) or P< 0.001(***). n.s., not significant. Analysis was performed by researcher blinded to the experimental conditions. (D) WT mice spent less time in the open arms. (E) KO mice spent more time in the closed arms. Date information: All mice were male, 8-12 weeks old. WT, n=12 mice; KO, n=12 mice. Error bars represent the means ± S.E.M.; Two-tailed unpaired t-test, P < 0.05 (*), P < 0.01(**) or P< 0.001(***). n.s., not significant. Analysis was performed by researcher blinded to the experimental conditions. (F-G) Representative motion trail from the Y-maze test. (H) Statistical analysis of the Y‐Maze test. S, O, and N represent the start arm, old arm, and new arm. The percent of time that mice stayed in each arm was recorded and analyzed. Whiskers represent the minimum to maximum value; central lines represent median values; boxes represent the values between upper quartile and lower quartile. Compared with those of WT mice, the entries of KO mice into new arms were decreased, while the entries into old arms were increased. (I) Impaired social communication, as demonstrated by ultrasonic vocalizations (USVs) in WT and KO pup mice. (J) Statistical analysis show that KO mice had with less call duration than WT. KO group were less frequent and the duration was significantly shorter than that of the WT group. n=9 mice, independent replicates. Date information: All mice were male, 8-12 weeks old. WT, n=12 mice; KO, n=12 mice. Error bars represent the means ± S.E.M.; Two-tailed unpaired t-test, P < 0.05 (*), P < 0.01(**) or P< 0.001(***). n.s., not significant. Analysis was performed by researcher blinded to the experimental conditions. (K) The total number of KO calls is less than that of WT calls. n=9 mice, independent replicates. Date information: All mice were male, 8-12 weeks old. WT, n=12 mice; KO, n=12 mice. Error bars represent the means ± S.E.M.; Two-tailed unpaired t-test, P < 0.05 (*), P < 0.01(**) or P< 0.001(***). n.s., not significant. Analysis was performed by researcher blinded to the experimental conditions. (L) Social interaction behavior is impaired in mH2A1.2 KO mice. WT mice spent similar time in the chamber with the stranger 1 or stranger 2 mice, while WT mice spent more time in the chamber with stranger 2 than with stranger 1. (M) Close social interaction is affected by mH2A1.2 deletion. KO mice did not spend more time in close interaction with stranger 2 than with stranger1 mice. Date information: All mice were male, 8-12 weeks old. WT, n=12 mice; KO, n=12 mice. Error bars represent the means ± S.E.M.; Two-tailed unpaired t-test, P < 0.05 (*), P < 0.01(**) or P< 0.001(***). n.s., not significant. Analysis was performed by researcher blinded to the experimental conditions.
Figure 4(A). Time course analysis of Xist expression by RT-qPCR during EB differentiation of female mESCs following knock down of Luciferase (Control, black) and Kap1 (Kap1 KD, grey), at the indicated timepoints. Data are presented as mean ±standard deviation from three independent experiments. Statistical significance was determined using 2-way ANOVA. Xist primers Xist ex3 F and Xist ex4 R were used. Normalisation was performed using a geometric mean consisting of the expression of Rplp0, Ubiquitin and Sdha. (*p ≤ 0.05). (B). RT-qPCR analysis of Xist expression levels during differentiation of the Fa2L cells, following expression of shRNA against Luciferase (Control, black) and Kap1 (Kap1 KD, grey), at the indicated timepoints. Mean ±standard deviation from a minimum of three independent experiments is presented. 2-way ANOVA was used to determine statistical significance. ns=not significant. ( (C). Tsix RNA levels in female mESCs infected with shRNA directed against Luciferase (Control, black) and KAP1 (Kap1 KD, grey), during differentiation. Mean ±standard deviation from four independent experiments are shown. Statistical significance was determined using 2-way ANOVA. (**p ≤ 0.01). Values have first been normalised to a geometric mean consisting of the expression of Rplp0, Ubiquitin and Sdha. (D). RNA FISH analysis of Tsix expression during differentiation of female mESCs expressing an shRNA directed against Luciferase (control) or against Kap1 (Kap1 KD). Left: Cells with no (0, red), one (1, grey) and two or more (2, black) Tsix foci were counted in two independent experiments, shown averaged. Statistical significance was determined by χ2. A minimum of 110 cells were counted per each time point for each line (**p ≤ 0.01, ****p ≤ 0.0001). Right: examples of cells with one (top) or two (central and bottom) Tsix FISH signals. Scale bars: 5μm.
Figure 6Number of mGFP and mTomato positive preadipocytes isolated by FACS from the indicated white adipose depots from 5-6 month old male Wt1-creERT2;Rosa26mT/mG, Tagln-cre;Rosa26mT/mG, and Mx1-cre;Rosa26mT/mG mice. Data are shown as mean ± SEM of 4-9 mice.
Figure 5A. Total Chol levels in WT and CCR5-/- OT-II lymphoblasts (day 10, IL-2-expanded) as determined by a fluorometric assay (n = 6). B-D. SM (B), Cer (C) and dhCer (D) levels in WT and CCR5-/- OT-II 10-day lymphoblasts, as determined by UPLC-TOF MS. Values, after normalization with C17 standards and cell number in each sample, are the mean of two independent experiments (n = 6). E. RT-qPCR determination of CerS mRNA levels in naïve and IL-2-expanded WT and CCR5‑/- OT-II 10-day lymphoblasts (n = 3-5). F. Representative immunoblot showing CerS2 protein levels in naïve and WT and CCR5-/- OT-II 10-day lymphoblasts, and densitometric quantification of blots as above (n = 10). G. ChIP analysis of the CerS2 promoter using an anti-H3K9Ac antibody. Scheme of the CerS2 promoter showing CpG islands and primers used for amplification. Relative ChIP of the CerS2 promoter in WT and CCR5-/- OT-II 10-day lymphoblasts (n = 3). H. Relative CerS2 mRNA level in CD4 T cells treated with PTx (n = 3). I. Scheme of a canonical CerS gene to illustrate the in silico strategy used to search for CerS-specific transcription factors. J, K. Venn diagrams showing the number of transcription factors with putative binding sites in the indicated CerS genes in regions 1 (J) and 2 (K). The red circle highlights the transcription factors shared by CerS2, CerS3 and CerS4 promoters, but not present in the CerS6 promoter. L. Representative immunofluorescence images showing pSer142-GATA-1 staining (green) of OT-II WT and CCR5-/- lymphoblasts. The green channel (top) and the merge with nuclear DAPI staining (blue; bottom) are shown. Scale bar, 10 μm. M. Quantification of nuclear staining of the cells plotted as integrated density fluorescence intensity in DAPI-stained area; n ≥50 cells/condition). N. Top, basic scheme of the CerS2 promoter, indicating the putative GATA-1 binding site (blue) and location of the primers used for amplification in ChIP assays (black arrows). Bottom, relative anti-GATA-1 ChIP levels in OT-II WT and CCR5-/- lymphoblasts (n = 5).
Figure 6A. Schematic of mass-spec (MS) proteome profiling. Cortex and OB were isolated from Daam2 Het and KO mice (N=3 each) and digested for peptide library generation. Total numbers of detected and differentially expressed proteins in each brain region are indicated in pie charts. Venn diagram shows differentially expressed genes in each region and genes differentially expressed in both. B. Gene ontology (GO) analysis of differentially expressed genes in each region. C. Heat maps of commonly regulated genes in both regions. Genes differentially regulated in opposite directions in the cortex and OB are marked with *. D. Heat maps of uniquely up- and down-regulated genes in the cortex and OB. E-F. qRT-PCR (E) and immunostaining (F) confirm differential expression of genes found in MS profiling. qRT-PCR is repeated as triplicate and is presented as mean ± SEM. *p<0.05, **p<0.01, ****p<0.0001. Student's t-test was used. Scale bar: 20 μm.
Figure 1A. Mutations in the kinase motif abolished the yeast toxicity of MavQ. S. cerevisiae was transformed with plasmids expressing Flag-tagged wild-type MavQ or the indicated MavQ mutants under the galactose-inducible promoter. Yeast cells were spotted on synthetic medium supplemented with glucose or galactose for 3 days before image acquisition. B. The expression of MavQ in the yeast was detected by Flag antibody. The PGK (3-phosphoglyceric phosphokinase) was used as a loading control.
Autophagy receptors p62 and NDP52 localize to distinct pools of capsid and have distinct effects on CHIKV infection in Hela cells.Figure 2Autophagy receptors p62 and NDP52 localize to distinct pools of capsid and have distinct effects on CHIKV infection in Hela cells. Cells were infected for 15 h and labeled using antibodies to NDP52 and capsid (A), to p62, NDP52 and capsid (B), to NDP52, LC3‐C or capsid (C), to p62, ubiquitin (FK2) and capsid (D), or to NDP52, ubiquitin (FK2), and capsid (E). Cells were treated with CTRL or p62 siRNA, then infected for 24 h. Cell mortality (that is, fold change relative to mock‐infected cells) (F), viral replication (left) and qRT‐PCR (right) (G), and viral production (H) were assessed and whole‐cell lysates were immunoblotted for p62, capsid or actin (I). Cells were treated with CTRL or NDP52 siRNA, then infected for 24 h. Cell mortality (that is, fold change relative to mock‐infected cells) (J), viral replication (left) and qRT‐PCR (right) (K) and production were assessed (L). Whole‐cell lysates of siRNA‐treated cells were immunoblotted for NDP52, capsid or actin (M). Images and graphs shown are representative of at least three independent experiments and data presented in graphs correspond to mean+s.d. (n=3). Scale bar, 10 μm. C, capsid; CHIKV, Chikungunya virus; CTRL, control; NDP52, nuclear dot protein 52 kDa; qRT‐PCR, quantitative reverse transcription polymerase chain reaction; siRNA, short interfering RNA.
Figure 2(A) At 48 hours post transfection of the indicated protease plasmids in triplicate, BHK21 cells were incubated with a fluorogenic substrate and wtSPINK6 or loss-of-function mutSPINK6 or PBS for 2 hours and then applied to fluorescence assay. Data represent mean and SD of triplicated wells in a representative experiment performed 3 times. **, P<0.01. Student's t test. (B) At 48h after co-transfection of the indicated protease plasmids and H7 or H1 plasmid, BHK21 cells were lysed to examine HA cleavage with WB. (C) At 48h after triple-transfection of H7, the indicated proteases and wtSPINK6 or vector, BHK21 cells were lysed for the detection of HA cleavage. (D) At 48h after triple-transfection of H7, HAT or TMPRSS2, wtSPINK6 or mutSPINK6, BHK21 cells were lysed for the detection of HA cleavage. (E) At 48h after triple-transfection of H1, the indicated proteases and wtSPINK6 or mutSPINK6, BHK21 cells were lysed for the detection of HA cleavage.
Figure 6A. Quantification of Fbxl17 mRNA levels in DAOY cells transfected with control shRNA or shRNA targeting Fbxl17. (mean ± SEM from 3 independent experiments, ***p<0.0005). B. Detection of Sufu protein levels in DAOY cells transfected with control shRNA or shRNA Fbxl17. Representative image of three independent experiments is shown. C. Quantification of Gli1 mRNA levels in DAOY cells transfected with control shRNA or shRNA Fbxl17. (mean ± SEM from 3 independent experiments, **p<0.005). D. T2- weighted magnetic resonance imaging (MRI) showing tumour development in rats injected with DAOY cells transfected with control shRNA or shRNA against Fbxl17. One representative image for each condition, at 10 weeks is shown. Scale bar: 5mm. E. Graph showing area of T2 hyperintensity (a surrogate marker of tumourgrowth) between 4 and 10 weeks post-tumour induction. Animals were injected with DAOY cells stably expressing control shRNA or shRNA against Fbxl17 (10,000 cells/1µL). (mean ± SEM; n = 5; two-way ANOVA, followed by unpaired t-test, *p<0.05, **p<0.01). F. Representative immunohistochemistry image of Vimentin staining. Scale bar: 1 mm.G. Quantification of tumour growth (vimentin staining) in rats injected with DAOY cells transfected with either control shRNA or shRNA against Fbxl17 (mean ± SEM; n = 5; unpaired t-test, *p<0.05). H. Representative immunohistochemistry image of Ki67 staining. Scale bar: 0.5 mm/0.1 mm.I. Quantification of proliferative index (Ki67 staining) in rats injected with DAOY cells transfected with either control shRNA or shRNA against Fbxl17 (mean ± SEM; n = 5; unpaired t-test, *p<0.05).