EMBO/sd-character-level-ner huggingface dataset:
Genetic and molecular features of XRCC4-mutant patients.Figure 1Pedigree. Black symbols designate affected subjects. Schematic view of wild-type and mutant (R225*) XRCC4 proteins with their main domains and phosphorylation sites. The numbering refers to amino acids of refseq: NP_071801.1. CTR: C-terminal region. Electropherograms of the XRCC4 genomic region encompassing the nucleotide substitutions in available members of the family. Quantitative real-time PCR of XRCC4 relative to GAPDH mRNA in affected (II-1 and II-2) and control (CT1 and CT2) fibroblasts. Each value refers to the mean of three independent experiments performed in duplicate. Error bars indicate the standard deviation. Immunoblot analysis of total lysates from fibroblasts (fbs) and of in vitro-synthesized proteins (i.v.p). fl: in vitro-translated full-length XRCC4 protein; ret.: reticulocyte lysate used for in vitro protein synthesis; 225*: in vitro-translated XRCC4 R225* truncated protein; Ct: lysate of control fbs; and II-1 and II-2: lysates of fbs from patient II-1 and II-2. α-XRCC4 (Abcam, 1:1,000) and α-GAPDH antibodies were used. Immunoblot analysis of total lysates from fibroblasts using α-LIG4 (GeneTex, 1:1,000) and α-SDHA antibodies. Ct: lysate of control fbs; II-1 and II-2: lysates of fbs from patient II-1 and II-2. The position of the molecular weight (MW) marker proteins is indicated.
Pharmacological clearance of senescent cells with Navitoclax (ABT 263) reduces cardiomyocyte senescence and stimulates cardiomyocyte regeneration.Figure 8Schematic depicting experimental design. Mice at 100 weeks (23-months) of age were treated with vehicle (Veh) or navitoclax (Nav) intermittently for 2 weeks. At 104 weeks mice were injected every day with EdU for 1 week. Quantification of mean number of TAF and % of TAF-positive CMs in 24 months old wild-type mice treated with vehicle or navitoclax (50mg/kg/day). Data are mean± SEM of n=5-8 mice per group. More than 100 CMs were quantified per animal. CM cross-sectional area in 24 months old wild-type mice treated with vehicle or navitoclax. (Left) Representative images of Sirius red staining and (right) % of fibrosis area in 24 month-old mice treated or not with navitoclax. MRI analysis of ejection fraction (EF%) in 24 month-old mice treated or not with navitoclax. Data are mean±S.E.M of n=6 mice per treatment group. left ventricle mass (LVmass) index in 24 month-old mice treated or not with navitoclax. Data are mean±S.E.M of n=6 mice per treatment group. % of ventricle wall thickness (%WTC) in 24 month-old mice treated or not with navitoclax. Data are mean±S.E.M of n=6 mice per treatment group. Examples of confocal microscopy images of CMs positive for CM marker troponin C (TropC), EdU, Ki67 and Aurora B from navitoclax treated animals. (Upper Right panel) white arrows identify two nuclei in the same CM that have incorporated EdU. (Lower Right panel) white arrow identify EdU expressing Aurora B symmetrically between two nuclei. Scale bars represent 20µm. Table summarising numbers of CMs quantified in (J). Quantification of EdU positive CMs (mono- or multi-nucleated) in vehicle and navitoclax treated animals. Data are mean±S.E.M of n=5-6 mice per group. % of Ki67 positive CMs in 29 month old INK-ATTAC mice treated with vehicle or AP20187. Data are mean±S.E.M of n=4-6 mice per group.
a, WT and Park2−/− mice were infected with wild-type M.f4a, WT and Park2-/- mice were infected with wild-type M. tuberculosis by aerosol and lung bacterial burdens were determined by plating (means ± s.d., n = 5 per group, *P < 0.02 by Student's t-test). b, Enumeration of liver and spleenc.f.u. from mice infected in a, 21 days post-infection (means ± s.d, n = 5 per group, *P < 0.03 by Student's t-test). c, Survival of M. tuberculosis-infected wild-type and Park2-/- mice (n = 10, **P < 0.001 by log-rank test). d, Immunohistochemistry staining of lung sections from infected mice21 days post-infection, scale bar, 50 µm. e, Immunohistochemistry staining of lung sections from a human patient with active tuberculosis. Scale bars, 500 µm (×4), 50 µm (×40). f, Confocal microscopy images of humanlung sections from e immunostained for M. tuberculosis and parkin; DNA was visualized using DAPI (4′,6-diamidino-2-phenylindole). Scale bars, 25 µm. g, Mice were infected with wild-type L. monocytogenes by i.p. injection and bacterial burdens in livers and spleens were determined by plating (means ± s.d., n = 7 per group, *P < 0.04 by Student's t-test).
Pbx1 controls the specification and survival of mDAn.Figure 3(A) To the left, representative immunofluorescence microscopy/confocal images showing that the number of TH+cells is reduced in Pbx1-/- embryos. Deletion of Pbx1 decreases the proportion of NURR1+neuroblasts that become TH+neurons at E12.5, indicating that Pbx1 is required for the proper maturation of NURR1+neuroblasts. To the right, quantification of these experiments. (B) Ectopic PBX3 is detected in all rostro-caudal levels of the VM in Pbx1-/- mice at E12.5, indicating a repressive function of PBX1 on Pbx3 expression. (C) Representative immunofluorescence microscopy/confocal images and quantification showing that the conditional deletion of Pbx1 and Pbx3 (cKO, Pbx1flox/flox;Pbx3-/-;Th-IRES-Cre-ERT) reduces the number of PITX3+cells in rostral VM levels, but not of NURR1+cells, when compared to double heterozygous at E14.5 (cHET, Pbx1flox/+;Pbx3-/+;Th-IRES-Cre-ERT). White boxes are magnified to the right. (D) At E18.5, a drastic reduction of TH+, PITX3+ and NURR1+cells is detected from anterior to posterior levels in cKO embryos, but not in cHET. Pyknotic NURR1+nuclei are observed in cKO embryos (arrowheads). White boxes are magnified to the right. (E) The total number of TH+ mDA neurons is dramatically reduced and there is an increase in aCASP3+ cells in cKO embryos compared to cHET mice at E18.5.
Figure 8 (A) The experimental scheme. B220+CD11c+NK1.1+cells derived from tumour-bearing mouse liver or lungs were primed with LiCM or tethered with recombinant FX, and then injected into tumour-bearing- or TCM-stimulated mice; 24 h after the injection, tumour cells were injected. Tissues were analysed 48 h after the tumour cell injection  (B) Tumour cell homing in the lungs pre-treated with LiCM-primed B220+CD11c+NK1.1+cells from tumour-bearing WT or FX−/− mouse liver. Representative photos of homing of rhodamine-labelled tumour cells (upper, scale bar, 20 μm). Number of homing tumour cells are shown (lower). Shown are averages (N = 60 sections, 5/group, all field count/section, 12 sections/sample) with SEM and Welch"s t-test  (C) Tumour cell homing in the lungs pre-treated with activated FX-overexpressed B220+CD11c+NK1.1+cells (FX-OE-HepELs) from tumour-bearing lungs. Number of homing tumour cells after an injection of lung HepELs or FX-OE-HepELs are shown. Shown here are averages (N = 24 sections, 4/group, all field count/section, 6 sections/sample) SEM and one-way ANOVA  (D) Model of B220+CD11c+NK1.1+HepEL functions in the pre- and post- metastatic phases. In the pre-metastatic phase, tumour-stimulating liver induces FX expression in B220+CD11c+NK1.1+cells, then, the cells move into lungs to eliminate focal fibrinogens (left panel). In the post-metastatic phase, if circulating tumour cells reach the fibrinogen area, B220+CD11c+NK1.1+HepELs attack tumour cells with IFNγ (right)
a) Δ133p53α expression is induced by cycloheximide (CHX).f1(a) Δ133p53α expression is induced by cycloheximide (CHX). MRC-5 fibroblasts (at population doubling levels (PDLs) 51) were treated with CHX (25 μg ml−1) for indicated time periods and examined in immunoblot for Δ133p53α and full-length p53. β-actin was a loading control for normalization. The relative expression levels of Δ133p53α and full-length p53 are shown below the images. (b) Δ133p53α expression is induced by phosphoinositide 3-kinase inhibitors. MRC-5 fibroblasts (PDLs 51) were treated for 4 h with LY303511 (10 μM), LY294002 (10 μM), Wortmannin (50 nM) or dimethylsulphoxide alone (as control) and examined in immunoblot for Δ133p53α and β-actin expression. (c) Diminished Δ133p53α at replicative senescence is restored by treatment with bafilomycin A1. The immunoblot analyses were performed in early-passage (Y) and replicatively senescent (S) human fibroblast strains MRC-5 and WI-38. The examined PDLs were 30 (Y) and 60 (S) for MRC-5; and 30 (Y) and 54 (S) for WI-38. Bafilomycin A 1 treatment was at 100 nM for 4 h. The relative expression levels of Δ133p53α, full-length p53 and p62/SQSTM1 (normalized with β-actin) are shown. Anti-LC3B antibody detected LC3-I and LC3-II, which was used in quantitative analysis shown in d. (d) Replicative senescence is not associated with enhanced autophagy in general. LC3-II expression levels were normalized with β-actin (data in c) and are shown in the bar graphs as relative values to replicatively senescent cells without bafilomycin A1 (S/−; defined as 1). The LC3-II levels in replicatively senescent cells were higher than those in early-passage cells whether in the presence or absence of bafilomycin A1, which may suggest increased synthesis of autophagosomes at replicative senescence. However, the differences in LC3-II levels between the presence and absence of bafilomycin A1 were similar in early-passage and replicatively senescent cells: in MRC-5, 1.9 for Y (2.1−0.2) and 2.0 for S (3.0−1); in WI-38, 1.6 for Y (2.2−0.6) and 1.6 for S (2.6−1). These data indicate that early-passage and replicatively senescent cells have similar rates of autophagic flux, that is, the number of autophagosomes that are delivered to lysosomes for degradation.
PTX3 expression is developmentally regulated and astrocyte dependent.Figure 1(A) ELISA assa from brain lysates showing higher PTX3 expression in late embryonic and early postnatal brain, and lower in the adult brain. Data are presented as mean±SEM. (A) ng PTX3/mg of total proteins, E15=5,702±0.365, P0=4.904±0.865, P7=3.818±0.530, P14=2.140±0.107, P28=3.125±0.525, P90=2.212±0.324. One-way ANOVA analysis of variance followed by post hoc Tukey test: E15 vs P14 **p=0.001, E15 vs P28 *p=0.022, E15 vs P90 **p=0.002; P0 vs P14 $p=0.013, P0 vs P90 $p=0.016 ; 4 animals for each time point (B) real time qPCR from brain lysates showing higher PTX3 expression in late embryonic and early postnatal brain, and lower in the adult brain. Data are presented as mean±SEM (B) qPCR fold change normalized on E15, E15=1.037±0.154, P0=0.711±0.094, P7=0.472±0.0524, P14=0.135±0.007, P28=0.168±0.020, P90=0.183±0.029. One-way ANOVA analysis of variance followed by post hoc Tukey test: E15 vs P7 ***p=0.0001, E15 vs P14 **** p<0.0001, E15 vs P2 ****p<0.0001, E15 vs P90 ****p<0.0001; P0 vs P14 $$$p=0.0007, P0 vs P28 $$p=0.001, P0 vs P90 $$p=0.002; 4 animals for each time point Quantitation of PTX3 levels in pure astrocyte and neuronal cultures. (C) ELISA assay performed on astrocyte and neuronal culture mediu Only astrocytes release PTX3 (ELISA, astro=3.699±1.129, neu=N.D., not detectable. 3 independent astrocyte cultures evaluated Data are presented as mean±SEM Quantitation of PTX3 levels in pure astrocyte and neuronal cultures (D) rea time qPCR performed on astrocyte and neuronal lysates qPCR astro=1.167±0.288; neu=0.148±0.057; Mann-Whitney test **p=0.0095. 2 independent neuronal cultures evaluated. Data are presented as mean±SEM
Increased formation of punctate structures containing GFP-NBR1 under heat stress.Figure 4(Top panel) Four-weeks old transgenic wild-type Col-0 (WT), atg7-2 and nbr1-1 mutant plants expressing GFP-NBR1 were treated with (45°C) or without (22°C) heat shock for 3 h and then placed at room temperature for 0.5 h. The leaves were visualized by fluorescence confocal microscopy of GFP signal. (Bottom panel) Numbers of punctate GFP-NBR1 spots per 10,000 µm2 section. Means and SE were calculated from three experiments. According to Duncan's multiple range test (P = 0.05), means do not differ significantly if they are indicated with the same letter. Bar = 20 µm.
Figure 7A Upper panel: Model prediction of Epo-dependent proliferation in mCFU-E and BaF3EpoR cells overexpressing SHIP1 or PTEN. Solid lines represent model trajectories. Lower panel: Experimental validation of PTEN and SHIP1 overexpression effects on Epo-dependent proliferation. Proliferation was assessed using [3H]-Thymidine incorporation 14 h (mCFU-E) or 38 h (BaF3-EpoR) after retroviral transduction with PTEN or SHIP1 construct for overexpression. Data represented as mean ± standard deviation, N=3. EC50 values are given. B Upper panel: Model prediction of proliferation upon combined inhibitor treatment and 5 U/ml Epo stimulation. Maximum proliferation was scaled to 1. Lower panel: Experimental validation of proliferation upon combined inhibitor treatment and 5 U/ml Epo stimulation. Proliferation was measured as cell numbers after 14 h (mCFU-E) or 38 h (BaF3-EpoR, 32D-EpoR) with Coulter Counter. Maximum was scaled to 1
Figure 6A) Overview of BioID technique used to determine TRAPP subunit most proximal to TBC1D14. D = denaturing contitions, ND = non-denaturing conditions B) Samples prepared according to (A) (8 × 107 HEK293A cells per transfection) were lysed in TNTE, split equally and denatured with 1% SDS (D) or not (ND) and subjected to 1 hour streptavidin resin pulldown at room temperature. The bound proteins were eluted in 2x Laemmli sample buffer plus 3 mM Biotin and immunoblotted for the indicated proteins. C) siRNA transfected control cells (RISC Free) or TRAPPC8 knockdown (TRAPPC8) (1x10cm plate per condition) were lysed in TNTE and used in pulldown assays with immobilised recombinant GST or GST-TBR. Bound proteins were subjected to immunoblot analysis using TRAPPC8, TRAPPC4 and tubulin antibodies. Ponceau staining was used to verify loading of the GST fusion proteins (GST-TBR indicated with arrowhead). The bar chart expresses the relative amount of TRAPPC4 isolated from siTRAPPC8 compared to siRISC Free control cells from three independent experiments, error bars are ± s.e.m. ** p<0.01, unpaired T-test D) HEK293T cells stably depleted of TRAPPC8 (shC8) or not (shCtrl) were transfected with constructs encoding GFP or GFP-TRAPPC3 and subjected to GFP Trap IP. Precipitated proteins were immunoblotted for GFP, TRAPPC4 and TRAPPC8. Bar chart: the amount of TRAPPC4 isolated was normalised to the amount of GFP-TRAPPC3 precipitated from three independent experiments and plotted ± s.e.m., ns = non-significant, unpaired T-test.
The disruptive activity of KSHV‐TK is dependent on FAK and paxillinImmunoblot analysis reveals that GFP‐KSHV‐TK WT, Y2F and Y3F but not GFP‐KSHV‐TK‐DEAD associate with FAK.Figure 6A Immunoblot analysis reveals that GFP‐KSHV‐TK WT, Y2F and Y3F but not GFP‐KSHV‐TK‐DEAD associate with FAK. B GFP‐KSHV‐TK does not displace paxillin or zyxin from focal adhesions (white arrowheads) or induce contraction in the absence of FAK. Scale bar, 20 μm. C GFP‐KSHV‐TK does not induce loss of zyxin and p‐Tyr from focal adhesions or contraction of HeLa cells depleted of paxillin. Error bars represent SEM from three independent experiments, n = 196 for mock siRNA and n = 229 for paxillin siRNA; ***P 0.001. Scale bar, 20 μm.
a, Fluorescence microscopy HeLa cells infected with Shigella and Listeria visualized with 4′,6-diamidino-2-phenylindole (DAPI) (pseudo-coloured red), and Salmonella carrying mCherry-expressing vector (red).f1a, Fluorescence microscopyHeLa cells infected with Shigella and Listeria visualized with 4′,6-diamidino-2-phenylindole (DAPI) (pseudo-coloured red), and Salmonella carrying mCherry-expressing vector (red). The cis-Golgi (green) was detected by α-GM130 antibodies. Scale bar, 10 µm. b, Fluorescence microscopy of HeLa cells transfected with either IpaJ or VirA. The cis-Golgi (GM130, green) and F-actin (red) are shown. Scale bar, 10 µm. c, Fluorescence microscopy of HeLa cells infected with indicated Shigella mutant strains. Scale bar, 10 µm. d, Percentage of HeLa cells with disrupted Golgi morphology in 100 cells infected with either wild-type Shigella (M90T) or the indicated mutants. Error bars, means ± s.e.m. calculated from three independent experiments. e, Number of recoverable c.f.u. per mouselung24 h after intranasal inoculation with 1 × 106 of wild-type M90T or each mutant Shigella strain as indicated. Limit of detection (LOD) for this assay is indicated by the dotted line. Error bars, geometrical means ± s.e.m. **P<0.001.
a) The effect of Atg14L knockout (mAtg14−/−) on Atg16L dot formation.figf4(a) The effect of Atg14L knockout (mAtg14−/−) on Atg16L dot formation. Wild-type (WT) and mAtg14−/− ES cells were cultured in nutrient-rich or starvation medium for 3 h. Cells were fixed and subjected to immunofluorescence with an anti-Atg16L antibody. Images of Atg16L staining (grey-scale) and Atg16L merged with Hoechst staining (colour) are shown.(c) Quantification of the number of Atg16L dots in a. The data are the mean ± s.d. of three independent experiments. (b) The effect of Atg14L knockout on LC3 dot formation. WT and mAtg14L−/− ES cells were cultured in nutrient-rich or starvation medium for 3 h. Cells were fixed and subjected to immunofluorescence with anti-LC3 antibody. Images of LC3 staining (grey-scale) and LC3 merged with Hoechst staining (colour) are shown. Scale bars, 20 μm. (d) Quantification of the number of LC3 dots in b. The data are the mean ± s.d. of three independent experiments. For each of the experiments in c and d at least 13 cells were counted.
ConclusionBAG3 associates with aggresomes in vivo.Figure 4(A) Spinal cord sections of mice overexpressing SODWT and SODG85R (disease end‐stage) were immunostained for BAG3 and SOD1. Arrows indicate SOD1‐ and BAG3‐positive aggregates in the neuropil. Scale bar, 20 μm. (B) Localization analysis of BAG3 and SODG85R in surviving spinal cord motor neurons fromSODG85Rmice at the time of disease symptom onset. Arrows indicate perinuclear aggresomes containing BAG3 and SODG85R. Scale bar, 10 μm. Magnific., magnification; SOD, superoxide dismutase; SODWT, wild‐type SOD.
A) 293 cells were transfected with bag1, bag3 or nonsense (nons) siRNAs, as indicated.Figure 2(A) 293 cells were transfected with bag1, bag3 or nonsense (nons) siRNAs, as indicated. After 48 h, cells were transfected with d2GFP expression plasmid together with half the amounts of the indicated siRNAs. After additional 24 h, levels of indicated proteins were detected by immunoblot analysis. (B) 293 cells were transfected with indicated siRNAs for 48 h followed by real‐time PCR analysis of BAG1 and BAG3 mRNA levels. Depicted is the mean relative expression ratio (log2) ±s.e.m. *P0.05 and ***P0.001 versus nons, n=3. (C) BAG1 knockdown was performed in 293 cells for 48 h. Thereafter, cells were transfected with GFP or GFP‐based UPS reporter genes as indicated. After additional 24 h, levels of GFP‐positive proteins were detected by western‐blot analysis using a GFP antibody. (D, E) Immunoblot analysis of indicated proteins from d2HEK cells treated with different concentrations of MG132 for 5 h (D) or transfected with indicated amounts of BAG1S plasmid for 24 h (E).
Figure 6Purified h-cGAS protein was precipitated with Ctrl beads or cGAMP beads and then immunoblotted. THP-1 cells were stimulated with biotin-cGAMP for indicated times and then whole cell lysates (WCL) were precipitated with streptavidin beads followed by immunoblotting. Immunofluorescent staining of cGAS (red) in THP-1 cells treated with FITC-ecGAMP (5 μg/ml) (green) or FITC-icGAMP (0.1 μg/ml) (green) for 4 h, nucleus in blue (DAPI). Scale bar, 10 μm. Purified h-cGAS protein was precipitated with beads coupled with cGAMP, c-di-GMP or c-di-AMP followed by immunoblotting. Binding curves of surface-immobilized 2'3'-cGAMP with His-cGAS at indicated concentrations. Vertical lines mark the start of association and dissociation phases of the binding events. The dashed lines are global fits to a Langmuir reaction model; global fitting parameters are listed in the table below the plot. (n=4 independent experiment). The small-angle X-ray scattering analysis of the full-length cGAS with cGAMP. The EOM fit of the measured SAXS data. The goodness-of-the fit χ2 = 1.1. Structural alignment of the representative structures from cGAS-cGAMP EOM analysis. cGAMP subunits are colored red and blue, respectively. Different conformations of the cGAS N-termini are highlighted in additional shades of red and blue. The Dmax distributions (the maximum distance within a particle) derived from the EOM analysis of the measured SAXS profile (pool - the white histogram, the selected structures - the black histogram). The distribution of the selected structures shows a bimodal behaviour with an average value of 166.9 Å.
Figure 7A, B. The impact of HUWE1 on H2AX phosphorylation and ubiquitination, under normal conditions (A) or upon induction of replication stress (2mM HU for 18h, or 2h after exposure to 40J/m2 UV). Control or HUWE1-knockout HeLa cells were analyzed. C. In vitro ubiquitination assay showing that recombinant HUWE1C-ter can mono-ubiquitinate H2AX. D. Wild-type, but not the PIP-mutant can correct the defective H2AX ubiquitination in HUWE1-knockout 293T cells. . E. Chromatin immunoprecipitation showing that H2AX binding to the common fragile site FRA3B is reduced in HUWE1-knockout 8988T cells. Cells were treated with 600nM aphidicolin for 24h. Binding was quantified relative to input material. Shown is the average of 4 experiments -/+ SD. P-value is 4.4x10-6. F. Western blots showing that several H2AX ubiquitin ligases participate in H2AX ubiquitination following replication stress. HUWE1, RNF168, and BMI1 were knocked-down in HeLa cells. The efficiency of the knockdown is shown in Fig EV4. Cells were treated with 600nM aphidicolin for 24h, 2mM HU for 24h, or analyzed 2h after exposure to 40J/m2 UV. G. Chromatin immunoprecipitation from HeLa cells, showing that knockdown of H2AX ubiquitin ligases can reduce H2AX binding to FRA3B. Cells were treated with 600nM aphidicolin for 24h. Binding was quantified relative to input material. Shown is the average of 3 experiments -/+ SD. The p-values shown indicate the statistical significance relative to siControl (0.0016, 0.0005, and 0.0009 respectively). H. Chromatin fractionation experiments showing that HUWE1-knockout HeLa cells, treated with 2mM HU for 24h, fail to efficiently recruit BRCA1 and BRCA2 proteins to DNA. In contrast, 53BP1 chromatin association is not induced by HU treatment, and not affected by HUWE1 knockout, and thus can serve as loading control. Shown are blots of whole cell extract (WCE) samples, representing input material, and of chromatin pellet samples.