EMBO/sd-character-level-ner huggingface dataset:
Figure 1(A Representative confocal images of HeLa and TRIM16KO cells treated with (A) MG132 (20 µM, 2h), scale bar: 7.5 µm. and the samples were processed for IF analysis with Ub and p62 antibody. (B Representative confocal images of control siRNA and TRIM16 siRNA transfected cells treated with (B) MG132 (20 µM, 2h), where IF analysis was conducted with Ub and p62 antibodies. (C The graph shows the the percentage of cells with Ub-p62 co-localized dots. Data from ≥10 fields (40X), n=3, Mean ±SD, **p < 0.0003, *p < 0.002 (Student's unpaired t-test). Data information: Unless otherwise stated, scale bar: 10 µm. D) Representative confocal images of HeLa and TRIM16KO cells treated with (D) H2O2 (200 µM, 2h) and the samples were processed for IF analysis with Ub and p62 antibody. E) Representative confocal images of control siRNA and TRIM16 siRNA transfected cells treated with (E) H2O2, (200 µM, 2h), where IF analysis was conducted with Ub and p62 antibodies. F) The graph shows the the percentage of cells with Ub-p62 co-localized dots. Data from ≥10 fields (40X), n=3, Mean ±SD, **p < 0.0003, *p < 0.002 (Student's unpaired t-test). Data information: Unless otherwise stated, scale bar: 10 µm. (G) Western blot (WB) analysis of detergent-soluble and -insoluble fractions of HeLa and TRIM16KO cells treated with MG132 (50 µM, 1h) and probed with indicated antibodies. (H, I) Densitometric analysis (using ImageJ) of WB normalized with α-Tubulin or Lamin-B1. Data information: Unless otherwise stated, scale bar: 10 µm. (J) Representative confocal images of HeLa and TRIM16KO cells treated with MG132 (20 µM, 2h) and processed for IF analysis with Ub and LC3B antibody. (K, L) WB analysis of LC3B levels in lysates of HeLa and TRIM16KO cells treated with (K) Bafilomycin A1 (300 nM, 3h) alone or (L) with MG132 (10 µM, 4h) as indicated. L.E, Low exposure; H.E, high exposure. (M) Representative confocal images HeLa and TRIM16KO cells treated with H2O2 (200 µM, 2h) and processed for IF analysis with Proteostat dye, Ub, and p62 antibody. To show the cellular details, the brightness of TRIM16KO cells images was increased more than control cells (N) Pictorial representation of data presented
Generation of nanobodies blocking the SARS-CoV-2 RBD:ACE2 interface.Figure 1Nanobodies (Nbs) are genetically engineered from heavy-chain only antibodies of alpacas. The interaction between the SARS-CoV-2 homotrimeric spike protein and angiotensin-converting enzyme (ACE) 2 can be blocked by receptor-binding domain (RBD)-specific Nbs in the monovalent or biparatopic format.
A model on the roles of RNF26 in virus-triggered induction of downstream genes.Figure 8
Figure 6A IL-6 secretion of the indicated cell lines (ABC-DLBCL in red, GCB-DLBCL in green) and primary patient material, as quantified by ELISA and normalized to cell number (shown per 1000 cells). n.d., not detectable. STAT3 phosphorylation as assessed by Western blotting (B, D) of the indicated ABC-DLBCL and GCB-DLBCL cell lines, with and without 1h of exposure to 50ng/ml hIL-6 (B,D). STAT3 expression levels are shown as Western blot control. STAT3 phosphorylation as assessed by flow cytometry (C) of the indicated ABC-DLBCL and GCB-DLBCL cell lines, Histograms in C represent STAT3 phosphorylation in the absence of exogenously added IL-6. E STAT3 phosphorylation as assessed by Western blotting of CD126-proficient (WT) or -deficient (ko) RC-K8 cells, with and without 1h of exposure to 50ng/ml hIL-6. F Kinetics of STAT3 phosphorylation as assessed by Western blotting, of the IL-6-expressing SOCS1wt and SOCS1mut cell lines OCI-Ly3 and SU-DHL-2, under 1, 6 and 24h exposure to an IL-6-neutralizing antibody (α-IL-6, anti-IL-6 neutralizing antibody). STAT3 phosphorylation and gp130 expression of two representative cases (G) assessed by immunohistochemistry. The specific pSTAT3 and gp130 signal is seen as brown (DAB staining), and counterstaining was performed using hematoxylin (blue nuclei). STAT3 phosphorylation is more common in biopsies with gp130 expression; the p-value was calculated by chi-square test. Scale bar, 100 μm. STAT3 phosphorylation and gp130 expression of two representative cases (G) and summarized for the entire cohort of 114 patients (H), as assessed by immunohistochemistry. The specific pSTAT3 and gp130 signal is seen as brown (DAB staining), and counterstaining was performed using hematoxylin (blue nuclei). STAT3 phosphorylation is more common in biopsies with gp130 expression
A mutant in the API propeptide causes accumulation of prAPI on a nonvacuolar compartment.Figure 3A mutant in the API propeptide causes accumulation of prAPI on a nonvacuolar compartment. (A) Spheroplasts from an ape1Δ strain containing a plasmid bearing P22L API were subjected to differential lysis in PS200 buffer. The Total (T) fraction (after differential lysis) either received no treatment, was treated with 50 μg/ ml proteinase K, or received proteinase K in the presence of 0.2% Triton X-100. (B) The supernatant (S200) and pellet (P200) fractions were collected after centrifugation at 5,000 g for 5 min. The P200 fraction was resuspended in 10% Ficoll, and vacuoles were isolated by flotation through 4% Ficoll. V, vacuole; I, 4/10% Ficoll interface; P, gradient pellet. Proteins were detected by Western blotting. The positions of prAPI, mAPI, and mPrA are indicated. (C) ape1Δ cells containing P22L API on a plasmid were pulse labeled for 5 min, harvested by centrifugation, and subjected to a nonradioactive chase in either nitrogen-containing (SMD) or nitrogen starvation (SD-N) medium. Samples were collected at the times indicated and immunoprecipitated with API antiserum. The resulting SDS-PAGE gels were quantified using a Molecular Dynamics STORM phosphorimager.
Unless otherwise stated, experiments were repeated three times; data shown are representative.figf1(a) Different VPS34 complexes were immunoprecipitated (IP) from MEFs in the presence (N) or absence (−A) of amino acids using the indicated antibodies and assayed for kinase activity (left, top panel). Inputs were immunoblotted using the antibodies indicated (left, lower panels). Quantification of VPS34 activity is from 3 biological repeats (right panel; data shown are mean+s.d.). AR, autoradiography. (b) Immunoprecipitation of three VPS34 complexes, normalized for VPS34, was performed using the indicated antibodies under nutrient-rich (N) or starvation (-A) conditions. VPS34-binding partners were analysed by western blotting. (c) ATG14L-containing VPS34 complexes were immunopurified from WT, ULK1−/− 2KD (ULK def.) and FIP200−/− MEFs, and measured for lipid kinase activity as in a. (d) VPS34 was immunopurified from WT, ULK-deficient and FIP200−/− MEFs. A kinase assay was performed as in a. (e) Immunoprecipitation of Beclin-1-containing VPS34 complexes from WT, ULK-deficient and FIP200−/− MEFs. A kinase assay was performed as in a. (f) LC3B puncta and PtdIns(3) P levels were analysed with anti-LC3B and the biotin-2XFYVE domain probe. Representative immunofluorescence images of LC3B and 2XFYVE domain binding are shown (scale bars, 10 μm). (g) Quantification of LC3B puncta from f. Details of quantification are provided in the Methods; Data shown in g-i are mean+s.d. from a minimum of 6 unique fields of view from a representative experiment (see statistical source data in Supplementary Table S1). (h) Total PtdIns(3) P was quantified from the experiment in f. Error bars were calculated as in g. (i) Quantification of PtdIns(3) P that co-localizes with LC3B on amino-acid withdrawal from the experiment described in f. (j) VPS34 activity with or without ULK1 overexpression was assayed. A representative experiment of four repeats is shown.
Figure 2(A) Serum NfL levels of ataxic SCA3 subjects significantly correlated with disease severity, as quantified by the Scale for the Rating and Assessment of Ataxia (SARA) score (r=0.43, p<.001, Pearson's correlation, two-tailed test). (B) The cross-sectional NfL levels also reflected longitudinal disease progression, as quantified by the annual SARA score change (available for 35 subjects). Boxes show the ranges between lower and upper quartiles, the central bands show the medians, and the whiskers show data within 1.5∙IQR of the median, with dots representing outliers. Subjects with high disease progression (annual SARA score increase ≥0.71 points/year, n=18, median split) had significantly higher serum levels of NfL than subjects with low disease progression (C) We modelled serum NfL levels (log-transformed) in SCA3 carriers (n=123) with the predictors age and ATXN3 CAG repeat length, their squares and all possible interactions (for details, see Results and Methods). The highly significant predictors (age, its square, and repeat length, and the intercept, all p<.001, explained variance: R2=0.37) were used to generate the diagram. For a given age, each increase in CAG repeat count was associated with higher NfL concentrations. The steepness of the slopes declined with increasing age. In controls (black, n=125), the relation between NfL level (log-transformed) and age was linear.
LOX expression is required for invasion in a mutant p53-driven model of PDAC.Figure 2PC-1 signature is predictive of metastatic disease in mouse models of pancreatic cancer. Log-transformed expressions of signature transcripts from mouse tumor microarrays were mean-centered across samples and scaled to unit variance. These values were then multiplied by the matching loading values from the PC-1 signature and summarized for each sample across all transcripts to yield the risk score for that sample. Inverted invasion assays were performed with PDAC tumor cell lines from KPC and KPflC mice. Tumor cell lines bearing mutant p53R172H (KPC) invade significantly further than tumor cells with deletion of 1 copy of p53 (KPflC) (P ≤ 0.01). Data are shown as the average of four wells + SEM. Introduction of shRNA targeting Lox into KPC tumor cells significantly inhibits invasion (P ≤ 0.01 by unpaired Student's t-test). Data are shown as the average of four wells + SEM. Introduction of exogenous LOX into KPflC tumor cells significantly promotes invasion (left panel, P ≤ 0.01 by unpaired Student's t-test). LOX expression was assessed by immunoblotting (right panel). Columns indicate the mean of four well and error bars indicate SEM. Integration of heterogeneous data sets identifies LOX as a therapeutic target in pancreatic cancer. Components of the hazardous PC-1 signature are overlaid with genes found to be overexpressed in a microarray and hits that cause a reduction in viable cell number in an RNAi functional screen. Overlap is shown as a proportional Venn diagram.
Figure 4A) Schematics of engineered FoxO3 constructs. KD measurements of the LxxIxE motifs fused to the C-terminus of FoxO3 were performed previously (Hertz et al., 2016; Kruse et al., 2018). B) The indicated YFP-FoxO3 constructs were transfected into HeLa cells, purified using YFP resin and PP2A-B56 binding determined by Western blotting. PP2A-A; scaffold subunit. C) - D) YFP-FoxO3 constructs were transfected into HeLa cells and lysates (pS413) or YFP purifications (pS253) were subjected to Western Blotting and probed with the indicated phosphoantibodies. Quantifications arise from five (pS413) or three (pS253) independent experiments. Mean and standard deviation shown in plots as black bars. E) Steady-state localization of the indicated YFP-FOXO3 variants in HeLa cells. Each data point represents quantification from a single cell. ­A representative result from at least three independent experiments is shown. At least 25 cells were counted per condition in the experiment shown. Scale bar is 10 μm. Mann-Whitney test significance values; ∗p < 0.02, ∗∗∗p < 0.0002. F) Dephosphorylation by the PP2A-B56α holoenzyme complex of substrates with increasing length between phosphorylation sites and binding motifs as depicted. Engineered substrates containing three TP sites were phosphorylated with radioactive ATP using Cdk1 and incubated with the PP2A-B56α holoenzyme. Removal of radioactive phosphate was monitored over time. Mean and standard deviation from 3 experiments is shown.
Tau inhibits mitophagy in N2a cells A Immunostaining for the mitochondrial marker ATPβ in N2a cells expressing mito-QC.Figure 1A Immunostaining for the mitochondrial marker ATPβ in N2a cells expressing mito-QC. Data information: Data are given as mean and SEM, * = p < 0.05, **= p <0.01, **** = p < 0.0001 for simple effects. Scale bar = 5 µm in (A) B N2a cells expressing mito-QC and myc-Parkin together with hTau-V5, hP301L-V5 or an empty vector control. Treatment with CCCP (8 µM, 17 h) induces mitophagy in control cells, as indicated by a decrease in the GFP/mCherry fluorescence ratio, but not in hTau or hP301L cells. Deconvolution (Classic Maximum Likelihood Estimation) was applied to images for display only. C Quantification of GFP/mCherry fluorescence intensity per cell. Data were analysed by 2-way ANOVA, showing significant main effects of CCCP treatment, F (1, 314) = 15.66, p < 0.0001, and tau expression, F (2, 314) = 12.26, p < 0.0001, and a significant interaction effect, F (2, 314) = 6.137, p = 0.0024, n = 40-73 cells/group. Data information: Data are given as mean and SEM, * = p < 0.05, **= p <0.01, **** = p < 0.0001 for simple effects. Scale bar = 10 µm D Cells expressing mito-QC and myc-Parkin together with hTau-V5, hP301L-V5 or an empty vector control, treated with a mixture of antimycin and oligomycin (AO, 15 µM, 17 h). E Quantification of GFP/mCherry fluorescence intensity per cell. Data were analysed by 2-way ANOVA, showing no significant main effects of AO treatment, F (1, 212) = 0.564, p = 0.4534, or tau expression F (2, 212) = 1.579, p = 0.2087, but a significant interaction effect, F (2, 212) = 9.854, p < 0.0001, n = 16-50 cells/group. Data information: Data are given as mean and SEM, * = p < 0.05, **= p <0.01, **** = p < 0.0001 for simple effects. Scale bar = 10 µm
Figure 2A ral hypomorph mutants (ral35d/Y;; UAS-p35/+), n= 20, animals with salivary gland-specific expression of p35 (ral35d/+;; UAS-p35/fkh-GAL4), n= 21, and ral hypomorph mutants with salivary gland-specific expression of p35 (ral35d/Y;; UAS-p35/fkh-GAL4), n= 20, were analyzed by histology for the presence of salivary gland material (red dotted circles) 24 hours after puparium formation.B Quantification of data from (A). Data are represented as means. Statistical significance was determined using a Chi-square test comparing the percentages of gland fragments. C Salivary glands dissected from 0 hour after puparium formation control animals (+/w; UAS-ralIR/+) and those with salivary gland-specific knockdown of ral (fkh-GAL4/w; UAS-ralIR/+) and stained with cleaved Caspase-3 antibody (green) and DAPI (blue). Scale bars represent 20µm.D Salivary glands dissected from 13 hour after puparium formation control animals (+/w; UAS-ralIR/+) and those with salivary gland-specific knockdown of ral (fkh-GAL4/w; UAS-ralIR/+) and stained with cleaved Caspase-3 antibody (green) and DAPI (blue). Scale bars represent 20µm.E Quantification of data from (C and D). Data are represented as means ± SEM; n≥ 10. Statistical significance was determined using a Student's t test.
Figure 6(A) Lung sections from vehicle and barasertib treated mice were immunostained using Ki-67 antibody. Representative images were obtained at 20X magnification. Scale bar: 50μm. (B) Immunoblot analysis in lung lysates from vehicle and barasertib treated mice using PCNA antibody. Gapdh is used as loading control. **P< 0.005, 1-way ANOVA, (n=4-5 mice/group). (C) Quantification of Aurkb, Plk1 and CcnA2 gene transcripts in total lungs of mice treated with vehicle or barasertib. **P< 0.005, ***P<0.0005, ****P < 0.00005, 1-way ANOVA, (n=8 mice/group).
Deletion of de novo DNA methyltransferases does not influence neurogenic activity of adult hippocampal NSPCs in vivo.Figure 4A, No difference in numbers of Ki67-positive, proliferating cells was observed between the subgranular zone (SGZ) of Dnmt3a/b-WT and Dnmt3a/b-KO mice one week after tamoxifen administration. Depicted are data points for every animal with genotype means ± SEM. B, Bright field image of Ki67-positive cells stained using diaminobenzidine method. Scale bar: 100 µm. C, KO of Dnmt3a/b did not affect numbers of new-born neurons in the dentate gyrus. Mice were administered with tamoxifen and analyzed 5 weeks after the first injection. BrdU was injected 3.5 weeks before analysis. Depicted are data points for every animal with genotype means ± SEM. D, Fluorescence image of BrdU/NeuN-positive new-born neurons. Scale bar: 25 µm. E, Experimental scheme for results depicted in Fig. 4F-I, Fig. EV3B-C and Fig. 5A-B. Recombined WT and KO cells were identified by Gfp expression in Nestin::Cre-ERT2 mice. F, Gfp-positive cells were isolated by FACS from WT and KO mice and their transcriptional profile analyzed using single-cell RNA sequencing (n = 4 mice per genotype). t-distributed Stochastic Neighborhood Embedding (t-SNE) clustered cells into 4 populations which were labeled with T1-T4 and highlighted by color. Clusters T1b and T4b were identified as sub-clusters of T1 and T4, respectively. G, Marker expression identified the cell clusters as distinct cell stages during adult hippocampal neurogenesis. H, Distribution of WT (red) and KO cells (violet) into the different cell clusters. I, Percentages of WT and KO cells in the different cell clusters relative to all sufficiently covered cells (in total n = 293 cells from 4 mice, WT; n = 344 cells from 4 mice, KO). Depicted are percentages of cells in cluster per animal (individual data points) with genotype means ± SEM.
a) Blood glucose and insulin levels measured in WT (n = 6), Nlrp3−/− (n = 5) or Pycard−/− (n = 6) mice under fasting or refed conditions after 12 weeks of HFD.figf8(a) Blood glucose and insulin levels measured in WT (n = 6), Nlrp3−/− (n = 5) or Pycard−/− (n = 6) mice under fasting or refed conditions after 12 weeks of HFD. (b-e) Glucose tolerance test (GTT) (b,d) and insulin tolerance test (ITT) (c,e) performed for WT, Nlrp3−/− (b,c) and Pycard−/− (d,e) mice on HFD for 12 weeks. (f) GTT conducted on the indicated bone marrow chimeric mice on HFD for 12 weeks. (g,h) Insulin-stimulated phosphorylation of IRβ, IRS1 and Akt (Ser473) in liver tissues of individual WT and Pycard−/− mice (g), and p-Akt (Ser473) in liver, white adipose (WAT) and muscle tissues of individual WT and Nlrp3−/− mice (h) on HFD for 12 weeks, after insulin (2 IU/kg) infusion. Graphs at right of blots show the quantitation of each molecule. pY, phosphotyrosine. (i,j) Expression of Tnfa and Mcp1 mRNA relative to Actb in liver tissues of Nlrp3−/− mice (i) and Pycard−/− mice (j) on regular diet (RD) or HFD for 12 weeks. One of two independent experiments is shown (mean ± s.d.). *P 0.05 versus controls.
Figure 5A Schematic of BSP constructs with eGFP fusions. B Representative fluorescence microscopy images of BSP and its subconstructs (20 μM) at varying pHs. PEG 8K concentration is 2 %. Scale bar, 20 μm. C Phase diagram of 20 μM proteins as a function of pH and PEG 8K concentration, which was scored by the presence or absence of droplets in the samples. D Time lapse FRAP of liquid droplets formed by fluorescently labeled BSP, BSP12 and BSP123. Bleaching was performed at the indicated time points after mixing, and the recovery occurring was recorded. Data are reported as mean ± SD. SD is depicted as shadows in a light color. n = 3 liquid droplets. n.s., not significant (P > 0.05); ***P < 0.001; [one-way analysis of variance (ANOVA)]. E Quantification of droplet area is shown for each protein under various Ca2+ concentration conditions. Each sample is quantified by total area (left) and mean area (right). Each experiment was repeated three times, data shown as mean ± SD.
Effect of RK-33 on radiation-induced DNA damage.Figure 9A Immunofluorescence images showing 53BP1 and γH2AX foci in A549 cells after 2-Gy radiation and A549 cells pre-treated with 6 μM RK-33, 12 h before radiation. Overlap of 53BP1 and γH2AX is seen in the merged picture of the co-immunofluorescence staining. Scale bar is 2 μm. B A549 cells were pre-treated with RK-33 and radiated with 2 Gy, and 53BP1 and γH2AX foci were counted as a measure of DNA damage. Cells with more than 10 foci 53BP1 or γH2AX were counted. More than 400 cells per sample were evaluated. C H1299 cells stably transfected with a homologous recombination (HR) reporter construct were treated with RK-33. Reporter constructs expressed GFP, which was quantified by flow cytometry. Experiments were repeated three times. D H1299 cells, containing a stable non-homologous end-joining (NHEJ) reporter construct, were treated with RK-33 and knockdown of DDX3. Reporter construct expressed GFP, which was quantified by flow cytometry. All experiments were repeated three times. E Microarray results from MDA-MB-231 cells treated with RK-33 and shDDX3 were validated by qRT-PCR using NHEJ Mechanisms of DSBs Repair PrimePCR plates (Bio-Rad) and performed in biological triplicates. F, G DNA repair-related proteins (ATR and XRCC4), DDX3, and actin were assessed by immunoblotting in A549 (F) and H1299 (G) cells. Cells were pretreated for 4 h with vehicle control or 6 μM RK-33 and then radiated with 0 or 5 Gy. Outlined boxes indicate spliced lanes.
CENP-A nucleosome has flexible DNA ends independently of the wrapped DNA sequence.Figure 1A. Graphs showing the relative abundance of undigested DNA as a function of time during digestion with micrococcal nuclease (MNase) for three types of DNA wrapped around the CENP-A nucleosome (red) and the H3 nucleosome (green). For α-satellite DNA with initial size of 171 bp, size ranges (141-171) corresponding to DNA lengths above NCP (nucleosome core particle) is presented. Data are presented as mean (SD) for each time point based on three independent experiments. B. CryoEM density map of the human CENP-A nucleosome, color-coded for histones and DNA. C. CryoEM maps of the H3 nucleosome (PDB 6ESF) and the CENP-A nucleosome, colored based on local resolution. D. Overlay of cryoEM maps of the H3 nucleosome (green; PDB 6ESF) and the CENP-A nucleosome (red). Note shorter and moved density for DNA on the CENP-A nucleosome indicated by the arrow. E. Overlay of the N-terminal tail of CENP-A (red) and H3 (green; PDB 6ESF), illustrating shorter αN helix of CENP-A (obstructed by the presence of bulky CENP-AW47) and DNA moved by 4 Å. F. The N-terminal tail of CENP-A (red) makes contacts with the DNA (cyan) at the entry/exit sites.
Figure 3(A) Mitophagic flux in cells transfected with siNDP52, siTBK1, siOPTN or a combination of siRNAs. Results are the summary of three independent experiments. Values are the means ± SEM. **p < 0.01 vs scrambled oligo-transfected cells; †p < 0.05 vs siNDP52-transfected cells; §p < 0.05, §§p < 0.01 vs siTBK1; NS: not significant, determined with one-way ANOVA followed by the Tukey-Kramer post hoc test (B) HeLa-GFP-Parkin cells transfected with siNDP52, siOPTN, siTBK1 or scrambled siRNA were incubated with 1 µM valinomycin for the indicated times. Cell lysates were subjected to SDS-PAGE or Phos-tag-PAGE prior to western blotting using the indicated antibodies. Data are representative of two independent experiments. The position of phosphorylated forms of NDP52 or OPTN are indicated by arrowheads (C) Images of TBK1 KD cells expressing mCherry-NDP52 or mCherry-OPTN under valinomycin treatment from Movie EV 5. Cyan, GFP-Parkin; magenta, mCherry-NDP52. Images are representative of two independent experiments. Scale bars, 10 µm (D) TBK1 KD and control cells expressed mCherry-NDP52. Lysates were subjected to immunoprecipitation using anti-RFP beads. Immunoprecipitates were analyzed by western blotting. Data are representative of three independent experiments