EMBO/sd-character-level-ner huggingface dataset:
TCR stimulation renders T cells resistant to pyroptosis (A) CD4 T cells were activated with human T activator CD3/CD28 beads (bead:cell ratio 1:5) for 4 days.Figure 5(A) CD4 T cells were activated with human T activator CD3/CD28 beads (bead:cell ratio 1:5) for 4 days. Controls were kept resting in the presence of IL-7 and IL-15. On day 4 cells were stimulated as indicated for 18 hours and cytotoxicity was assessed by LDH-activity in the supernatant. Individual data points ± SEM from four independent donors. Statistics indicate significance by two-way ANOVA: ***p ≤ 0.001; ns, not significant. P-values were corrected for multiple comparisons (Sidak). (B) Immunoblotting of CD4 T cell lysates treated as in (A) for 3 to 4 days. * indicates an unspecific band. (C) Quantification of immunoblots shown in (B) and additional donors (n= 9 for all but GSDMD, GSDMD n=8, for one donor quantification of the GSDMD band was considered unreliable due to uneven substrate distribution). Individual data points ± SEM are shown. Statistics indicate significance by one-way ANOVA of log2-transformed fold change values of band intensities (activated/non-activated T cells) normalized to β-Actin: ***p ≤ 0.001; *p ≤ 0.05; ns, not significant. P-values were corrected for multiple comparisons (Sidak). All band intensity ratios were tested against β-Actin. CARD8 full length was additionally tested against CARD8 cleaved.
Figure 2(A-B) The effect of stable knockdown CSAG2 in HCT116 (A) or A375 (B) were assayed for clonogenic growth on plastic for 7 days. The number of colonies was quantified and is shown. Data are represented as the mean ± SD, n=3 biological replicates. (C-D) The effect of stable knockdown CSAG2 in HCT116 (C) or A375 (D) were determined for anchorage-independent growth in soft agar colony formation assays for 14 days. The number of colonies was quantified and is shown. Data are represented as the mean ± SD, n=3 biological replicates. (E-F) Knockdown of CSAG2 in A375 (F) or HCT116 (E) cells decreases xenograft tumor growth in mice. Cells with doxycycline-inducible shRNAs were implanted into NOD SCID gamma mice. Doxycycline (2 mg/mL) was administered via drinking water. Tumor growth was monitored over time. Data are represented as the mean ± SD, n=6 mice for each group. (G-H) Stable expression of CSAG2 in HCT116 (G) and CSAG2-negative H460 (H) cells increases anchorage-independent growth in soft agar colony formation assays, n=3 biological replicates. Data are represented as the mean ± SD.
Figure 4In the siRNA-transfected MCF7 (left) or T47D cells (right), the interferon signaling pathway was blocked at different stages by means of IFNβ neutralization The expressions of the ISGs were measured by qPCR. Each sample has 3 replicates. Error bars represent mean ± SD. In the siRNA-transfected MCF7 (left) or T47D cells (right), the interferon signaling pathway was blocked at different stages by means of , IFNAR2 neutralization The expressions of the ISGs were measured by qPCR. Each sample has 3 replicates. Error bars represent mean ± SD. In the siRNA-transfected MCF7 (left) or T47D cells (right), the interferon signaling pathway was blocked at different stages by means of JAK-STAT inhibition (C). The expressions of the ISGs were measured by qPCR. Each sample has 3 replicates. Error bars represent mean ± SD. Growth curves of the MCF7 (D) or T47D cells (E) upon HNRNPC knock-down but with the interferon response blocked with the IFNβ antibody. Each sample has 3 replicates. Error bars represent mean ± SD. Growth curves of the MCF7 (left) or T47D (right) cells upon HNRNPC knock-down and JAK-STAT inhibition with Ruxolitinib (5uM). Error bars represent mean ± SD. siNC: non-targeting siRNA as a negative control, siHN-1: siRNA sequence 1 for HNRNPC, siHN-2: siRNA sequence 2 for HNRNPC. Each sample has 3 replicates. Error bars represent mean ± SD.
Figure 4Heatmaps of gene expression in PLP-150Q mouse corpus callosum at 5 months after LAQ (5 mg/kg) treatment for 2 months revealed 27 significantly upregulated and 103 significantly downregulated genes. Most significant gene networks of different functions via GO-cluster and categorization. The bold-type indicated two phosphorylation associated clusters of "REGULATE_OF_PHOSPHORUS_METABOLIC_PROCESS", and "POSITIVE_REGULATE_OF_PHOSPHORUS_METABOLIC_PROCESS" The individual networks of up-regulated gene were heat mapped typically, on the clusters of "REGULATE_OF_PHOSPHORUS_METABOLIC_PROCESS", and "POSITIVE_REGULATE_OF_PHOSPHORUS_METABOLIC _PROCESS" Identification of consensus sequences from PRKG2 kinase substrate peaks, in which the same motif was identified using the 15 amino acid sequences surrounding the strongest 60 peaks. The PRKG2 substrate core consensus sequences of (R/K)2-3-X-S/T-X, where S=serine, R=arginine, K=lysine, T=threonine, X standing for any random amino acid, are mostly consistent with MYRF amino acid 252-266. The rat, monkey and human MYRF have the highly conserved core sequences of "KKRKHSE". Western blotting showing down-regulation of PRKG2 in PLP-150Q mouse corpus callosum at 3 months of age by 5 mg/kg LAQ. Ratios of PRKG2 or phosphorylated VASP (p-VASP) to GAPDH obtained from 3 independent experiments were presented on the right. One-way ANNOVA with Tukey's test. PRKG2: **P=0.0076(left) and ***P=0.0015(right); p-VASP: ***P=3.86x10-5(left) and 1.01x10-5(right). Data are mean ±SEM. Quantitative PCR of PRKG2 or PRKG1 gene expression in PLP-150Q mouse corpus callosum at 5 months of age after LAQ (5 mg/kg) treatment for 2 months. Only PRKG2 was decreased by LAQ. n=3 mice in each group. Student's t-test; **P=0.0073; ***P=0.00041. Data are mean ±SEM.
Figure 1Electron microscopy showing two adjacent mitochondria (one intact and one ruptured) in a DM platelet. a. Lower powered view of the single DM platelet. b. The enlarged inset highlights the ruptured mitochondria (with loss of cristae morphology) next to the intact mitochondria. c. Black dash line indicates mitochondria membrane and red dash line indicate phagophore formation. (M- mitochondria) Immunoprecipitation (Meng et al) of MsrB2 in HC and DM human platelets followed by MsrB2 and LC3I/II western. LC3 and MsrB2 immuno-EM analysis of HC and DM platelets. 15 nm dots indicate immunogold-labeled LC3 clusters and 5nm dots indicate immunogold-labeled MsrB2 clusters. No significant clusters were found in HC (a) platelets. Representative areas of clusters of gold labeling in DM patients (b-d) are presented. The nonparametric t test was performed for comparisons of 2 groups. Analysis was performed with Prism software (GraphPad Software, Inc, La Jolla, CA). A difference of P<0.05 was considered significant.
Figure 1A) Differences in baseline performance and rate of decline on each clinical measure for each participant; the heatmap indicates each participant's standard deviation (SD) from the group mean. B) Spearman's correlations between baseline performance and rate of decline for all clinical measures. C) Allele dosage or binary status for each genetic variable for each participant.
Figure 4(A-B) STK38 is required for XPO1_S1055 phosphorylation. (A) HeLa cells were transiently transfected with the indicated siRNA and subjected to Flag-XPO1(wt) transient transfection the following day. 48 hours later, cells were incubated with Okadaic Acid (OA, final concentration = 1 µM) for 1 hour or with DMSO. Immunoblotting was performed on whole cell lysates with indicated antibodies. (B) Graphical representation of the phospho S1055-XPO1 signal on total XPO1 (n = 3 independent experiments, mean ± SEM; *p<0.05, ns, not significant, Mann-Whitney test). As expected, XPO1 is phosphorylated on its Ser1055 upon OA treatment but not when STK38 is silenced (C) STK38 kinase activity is required for XPO1_S1055 phosphorylation. HekRasV12 cells silenced (or not) for endogenous STK38 were transfected with Flag-XPO1(wt) expression plasmid in addition to myc-STK38(wt) or myc-STK38(K118R) (STK38 kinase-dead version) plasmids. The next day, cells were treated with 1 µM OA or with vehicle (DMSO) for 1 hour. Flag fusions were immunoprecipited and pulled-down proteins were analyzed by western blotting. Upper panel displays immunoprecipited proteins and lower panel represents whole cell lysates.
GlcNAc affects ubiquitination of GAS.Figure 2A Expression levels of GlcNAc in WT GAS, ΔgacI, and ΔgacI::gacI were measured as the sWGA fluorescence intensity (normalized to WT GAS). B, C Accumulation of LC3-II. (B) Representative western blot of LC3-II during non-infection (NI), WT GAS, ΔgacI, and ΔgacI::gacI. (C) LC3-II intensity normalized to NI. D, E Recruitment of ubiquitin in GAS-infected cells. HeLa cells were infected with WT GAS, ΔgacI, and ΔgacI::gacI for 4 h, fixed, and immunostained for ubiquitin (FK2: magenta). Cellular and bacterial DNA were stained with DAPI (cyan). (D) Representative confocal images and (E) percentage of cells with ubiquitin-positive GAS. Scale bar, 10 μm. F Immunoblot of the indicated GAS isolated from host cells. Lysate of GAS-infected HeLa cells were precipitated with anti-lipoteichoic acid antibody to isolate GAS, and the immunoprecipitates were analyzed by immunoblotting. G, H Localization of ubiquitin in sWGA-treated GAS. HeLa WT were infected with the indicated GAS strains masked with sWGA for 4 h, fixed, and immunostained for ubiquitin (magenta). (G) Representative confocal images and (H) percentages of cell with ubiquitin-positive GAS. Scale bar, 10 μm. I, J Recruitment of p62 to gacI mutants. HeLa cells were infected with WT GAS, ΔgacI, and ΔgacI::gacI for 4 h, fixed, and immunostained for p62 (magenta). Cellular and bacterial DNA were stained with DAPI (cyan). (I) Representative confocal images and (J) percentage of cells with p62-positive GAS. Scale bar, 10 μm. K Bacterial viability rates of GAS mutants in cells (CFU recovered at 6 h post-infection/CFU at 2 h post-infection).
Figure 2(A) Light sensitivity: Photocurrents of a ReaChR-expressing RGC stimulated with varying light intensities at 590 nm (~1013 − 1018 photons cm-2 s-1). (B) ReaChR-induced photocurrents as a function of light intensity (590 nm, n = 7 cells, mean photocurrent amplitude at 4.2 × 1017 photons cm-2 s-1 was −349 ± 192 pA). At the bottom, the sensitivity range for wild-type cones (Nikonov et al, 2006) is shown in comparison to the sensitivity range of ReaChR expressing RGCs. The dashed blue and orange line indicate the maximum light intensities allowed in the human eye at 470nm and 590 nm, respectively (EU-Directive, 2006; ICNIRP, 2013). (C) Photocurrent action spectrum of a ReaChR-expressing RGC (1.2 × 1016 photons cm-2 s-1). (D) Normalized action spectrums of ReaChR-RGCs. Solid line represents patch-clamp data (n = 8 cells; 1.2 × 1016 photons cm-2 s-1). The mean photocurrent amplitude at 550 nm was −324 ± 97 pA. Dashed line represents multi-electrode array data (n = 168 cells; ~1016 photons cm-2 s-1). (E) Temporal properties. Modulation of ReaChR-induced photocurrents at increasing stimulation frequencies (patch-clamp recording from a ReaChR-expressing RGC; at 22 Hz magnified trace is shown; 1.3×1016 photons cm-2 s-1). (F) Spiking modulation ratio of ReaChR-RGCs quantified from multi-electrode array demonstrates reliable spike triggering at frequencies up to 30 Hz (297 cells, 1.3 × 1016 photons cm-2 s-1).
Figure 1A) U2OS cells were harvested at the indicated time points after treatment with ionizing radiation (15 Gy) followed by analysis by SDS-PAGE and immunoblotting with antibodies recognizing PALB2 and Vinculin. (NT, no treatment). The lysate from the 24 hours time point was treated with phosphatase (Ppase). B) U2OS cells were left untreated or treated with ATM (KU 55933, 10 μM), ATR (ETP-464, 1 μM), or DNA-PK (NU7441, 1 μM) inhibitors 30 minutes prior to exposure to IR (15 Gy, 2h recovery). Immunoblots were performed with indicated antibodies. C) U2OS cells were left untreated (NT) or exposed to HU (2 mM) for the indicated times. The lysate from the 24 hour time point was treated with phosphatase (Ppase). Immunoblots were performed with indicated antibodies. D) Untreated U2OS cells or cells exposed to either HU (2 mM, 24h) or IR (15 Gy, 2h recovery) alone or together with ATM inhibitor (KU55933, 10 μM) or inhibitors to ATR (ATR#1: ETP-464, 1 μM; ATR#2: AZ-20, 3 μM) were harvested and lysates were prepared. The inhibitors were added 30 minutes prior to HU or IR treatment. Immunoblots were performed with indicated antibodies. E) ATR kinase assay was performed with N-terminal PALB2 (aa 1-560; GST-PALB2-N) as a substrate and HA-tagged ATR as kinase and visualized by Fujifilm Phosphoimager. 293T cells transfected with empty vector (EV), GST, and ATR inhibition (AZ-20, 30 μM) are controls. HA-ATR and GST-PALB2-N was controlled by immunoblotting with HA and GST antibodies.
Figure 5A. Structure of the H4 N-terminal tail with corresponding cryoEM density in the CENP-A nucleosome (left) and CENP-A/CENP-CCR complex (right). Position of the H4 N-terminal tail within the nucleosome is boxed on ribbon diagram below. Histones and DNA are color-coded as in Figure 1 and cryoEM density assigned to CENP-C is shown in magenta. B. Overlay of the H4 N-tail from the CENP-A/CENP-CCR complex (blue) with the H4 N-terminus from the H3 nucleosome (green; PDB 6ESF) and H3CATD nucleosome (grey; PDB 5Z23).
Figure 3(A) Alternative splicing of sams-3, sams-4 and sams-5 in smg-2 (yb979) and smg-2 (yb979); sams-5 (gk147); sams-1 (ok2946) mutants. Synchronized L1 larvae of each strain were incubated in S-complete medium alone (lanes 1 and 5), with OP50 (OD600=10.0) (lanes 2 and 6) and with OP50 supplemented with 25 mM L-Met (lanes 3 and 7) or 25 mM cycloleucine (cLeu) (lanes 4 and 8) for 3 hours at 20°C. The splicing patterns were analyzed and presented as in Figure 2B (n=3). (B, C) Western blot analysis of SAMS-1, SAMS-3 and SAMS-4 during larval development in the smg-2 (yb979) (B) and wild-type (C) backgrounds. Genotypes of the worms are smg-2 (w) and smg-2; sams-5; sams-1 (s) in (B) and wild-type (w) and sams-1 (s) in (C). Synchronized L1 larvae of each strain were incubated with OP50 at 20°C and subjected to Western blot analysis at indicated time points. Anti-β-tubulin (B) or coomassie brilliant blue (CBB) staining (C) was used as a loading control. Specificity of the antibodies is confirmed in Appendix Figure S12. Note that upregulation of SAMS-1 protein in the wild type during larval development in (C) is consistent with feeding-induced upregulation of sams-1 mRNA in Figure EV3D.
Figure 6Wild type Balb/c mice were injected intra-peritoneally with solvent (daily) or 3 mg/kg chlorambucil (daily for 5 days) or 3.3 mg/kg cisplatin (daily for 3 days). Uptake of the apoptosis tracer 99mTc-Duramycin 2 h after intra-venous injection was quantified in selected organs using SPECT imaging in the indicated organs. Representative maximum intensity partial projections showing tracer distribution are shown. Immunohistochemical analyses of γH2AX staining in organs from mice treated as in A. Scale bar, 25 µm.
Figure 1(A) ANAC032expression in WT plants sprayed with Pst, 6 and 24 hpi, compared to control (sprayed with 10 mM MgCl2 (Mock)). Error bars represent means ± SD (n = 3; 'n' represents independently performed experiments, each including the rosette leaves of at least three plants grown in individual pots). ANAC032expression in WT treated with (B) SA or MeJA or (C) COR for 3 and 6 h compared to non-treated controls. In B and C, means ± SD are given (n = 3; 'n' represents independently performed experiments, each including at least 20 seedlings). FCh, fold change. Asterisks indicate a significant difference from their respective controls (p < 0.01; Student´s t-test).
Figure 2Representative image of comet assay for DNA fragmentation and the quantification of cells with comet tails in the regions of frontal cortices from 6-mon-old WT and FTLD-TDP Tg mice. Scale bar: 50 μm. n = 9 sections per mouse, N = 5 mice per group, data are presented as mean ± SEM, *p = 0.0114 by t-test. Representative IF staining of γH2AX and TDP-43 in the regions of frontal cortices from 6-mon-old WT and FTLD-TDP Tg mice. Nuclei were stained with DAPI (upper panel in blue) or NeuN (middle panel in green). Scale bar: 50 μm. The circled area is emphasized for showing the distribution of immunoreactivity in cell subregions. Scale bar: 15 μm. Lower panel: quantification of cells or neurons with γH2AX immunoreactivity and TDP-43 proteinopathies from each view of microscope. n = 9 sections per mouse, N = 5 mice per group, data are presented as mean ± SEM, ***p = 0.0008 by t-test. Representative IF staining of γH2AX and Ki67 in the regions of frontal cortices from WT and FTLD-TDP Tg mice. Nuclei were stained with DAPI (upper panel) or NeuN (middle panel). Scale bar: 50 μm. Subregions, scale bar: 15 μm. Lower panel: quantification of cells or neurons with γH2AX and Ki67 immunoreactivity from each view of microscope. n = 9 sections per mouse, N = 5 mice per group, data are presented as mean ± SEM, ***p = 0.0004 by t-test.
Figure 1A. Schematic of the predicted domain architecture of Chm7 and truncations. Green circle is a Chm7-specific insertion (relative to Vps25; see Appendix Fig S1). MIM is microtubule interacting motif. Red stripes denote putative autoinhibitory helix. Numbers are amino acid residues. B. Deconvolved fluorescence micrographs of Chm7-GFP and truncations with Nup170-mCherry NE marker and merge of green and red channels. Scale bar is 5 μm. C. Scatter plot of the mean fluorescence intensity with SD of individual NE foci (horizontal line is mean). >50 foci were measured for each GFP fusion; p values from un-paired student's T-test. *** is p ≤ 0.001; **** is p ≤ 0.0001. D. Deconvolved fluorescence micrographs of Chm7-GFP and Mps3-mCherry with merge of green and red images (right). Scale bar is 5 μm. E. 10-fold serial dilutions of chm7Δapq12Δ strains grown at the indicated temperatures with either an empty pRS416 plasmid (-) or those expressing the indicated Chm7 constructs (pRS416-HA-CHM7, pRS416-HA-chm7-NTD, pRS416-HA-chm7-CTD). See Appendix Figure S2A for protein levels.
Figure 1(A, B) Airyscan confocal analysis of PC1 (568, red) co-localization with GFP-LC3 (green) in (A) MEF (B) Saos2. Scale bars = 10 µm. (C) Airyscan confocal analysis of PC2 (647, red) co-localization with LC3 (488, green) in RCS cells. Scale bars = 10 µm. (A-C) The insets show higher magnification (A = x4.68; B = x6.76; C = x7.33) and single colour channels of the boxed area. (D) Quantification of GFP (A,B) or LC3 (C) vesicles positive for PC1 or PC2, expressed as % of total LC3 (mean +/- SEM), n = 18 cells (MEFs and Saos2); n=12 (RCS) from 3 independent experiments. (E-G) Scanning confocal microscopy analysis of MEFs, Saos2 and RCS cells treated with BafA1, immunolabeled for PC1 or PC2 and LAMP1. Nuclei were stained with Hoechst. (E, F) Scale bars = 10 µm, (G) Scale bars = 5 µm. The insets show higher magnification (E = x4.99; F = x6.49; G = x2.01) and single colour channels of the boxed area. (H) Transmission EM analysis in Saos2 cells, treated with BafA1, showing in detail a lysosome which contains immunolabeled PC1 (with nanogold particles), as indicated by arrows. (I) Scanning confocal microscopy analysis of Saos2 WT and CRISPR-Cas9 IDUA Saos2 at steady state, immunolabeled for PC1 and LAMP1. Nuclei were stained with Hoechst. Scale bar = 10 µm. The insets show higher magnification (left = x3.09; right = x3.12) and single colour channels of the boxed area. Bar graph shows quantification of lysosomes containing PC1 expressed as % of total LAMP1 per cell (mean +/- SEM). n= 31 WT cells, n=33 CRISPR cells counted; 3 independent experiments. Student's unpaired, two-tailed T-Test *** P<0.0001. (J) WT and CRISPR IDUA Saos2 lysed and analysed by western blot. Data are representative of 3 independent experiments.
Figure 8A) futsch genomic region (X1,408,435 - 1,454,00) and corresponding browser tracks from miCLIP-seq experiments in S2R+ cells. futsch transcripts contain two m6A peaks in its 5´ UTR, as indicated. B) Schematic f GFP reporter constructs containing an actin promoter and either wildtype 5´ UTR region of futsch or a mutated version (futsch**) containing two-point mutations (A - T) at the identified m6A sites. mCherry is under the control of a same actin promoter. (C, D) RNA immunoprecipitation assay. Quantification of GFP RNA levels upon immunoprecipitation of Myc-tagged Fmr1 or Flag-tagged Ythdf. Bars show average ± SD of technical triplicates (C) or Flag-tagged Fmr1 in the presence of overexpressed HA-tagged Ythdf. Bars show median ± S.E.M of biological triplicates. P-values were determined with an unpaired, two-tailed Student's t-test. (n.s. = not significant; p<0.01 = **) (D). Rpl15 levels were used as a normalization control and the enrichment of mCherry is shown as a negative control for background binding. E-G) Representative Western blot analysis of the protein level (E) and quantification of the translation efficiency of S2R+ cells transfected with the wildtype or mutated futsch reporter constructs upon control or Mettl3 KD (F) or Ythdf KD (G). LacZ dsRNA was used for the control KD. The translation efficiency was calculated as the ratio of the relative GFP protein level and GFP mRNA level. mCherry served as a normalization control for both the protein and mRNA level. Bars show mean ± s.e.m of biological triplicates. P values were determined with a Student's t-test. (n.s. = not significant; p<0.05 = *; p<0.01 = **). H) Model of the interplay between Ythdf and Fmr1 n translation control.
Figure 2A. Representative images of GFAP immunostaining in Daam2 cKO mice compared to controls in cortex and OB at P28. Scale bar: 20 μm. B. Quantification of GFAP immunostaining in Daam2 cHet and cKO mice from P3 to adult. Data is presented as mean ± SEM. n=6-17 images from N=4-6 mice per each genotype and age. Student's t-test is used for statistics. *p<0.05, ***p<0.001, p****<0.0001. C. Quantification of GFAP immunostaining in Daam2 cHet and cKO mice in cortex and OB at P28. Data is presented as mean ± SEM. n=17-33 images from N=5 mice per each genotype and age. Student's t-test is used for statistics. p****<0.0001. D. Western blot analysis showed a significant increase in the amount of GFAP protein in Daam2 KO primary astrocytes. N=2 E. Schematic of virus injection to overexpress Daam2 in the cortex. F. Overexpression of Daam2 via injection of AAV-GfaABC1D-myc-Daam2 in wild type P1 pups revealed reduced GFAP intensity compared to animals injected with control virus (AAV-GfaABC1D-GFP). AAV-GfaABC1D-myc-Daam2 was injected into Aldh1l1-GFP reporter mice for morphological complexity analysis and infected cells (Myc positive) were compared with non-infected cells. Dotted white line indicates virus-infected cells. Filled triangles and empty triangles indicate astrocytic primary branches with the control and reduced level of GFAP or Aldh1l1-GFP, respectively. Scale bar: 20 μm. G. Quantification of GFAP immunostaining in control virus-positive cells and Daam2 overexpressing cells. Data is presented as mean ± SEM. n= 22 images from N=3 mice per genotype. Relative fluorescent intensity was quantified, and Student's t-test was used, **** p<0.0001. H-I. Sholl analysis (H) and quantification of total length (I) of filament with overexpression of Daam2. Data is presented as mean ± SEM. n=17-32 images from 3-4 mice per each. **p<0.01, ***p<0.001. Two-way ANOVA and Student's t-test was used for H and I, respectively.