EMBO/sd-character-level-ner huggingface dataset:
Role of SlHAK20 in the salt-tolerant tomato.Figure 4A Generation of mutations in SlHAK20Hap1 by CRISPR/Cas9 using two independent single-guide RNA (sgRNA1 and sgRNA2). Sequences of SlHAK20Hap1 in wild type (TS-21), slhak20-1 and slhak20-2 mutant tomato are shown. The sgRNA-targeted sequences are underlined and the protospacer-adjacent motif (PAM) sequences are in blue. The deletion is indicated by a dashed line and the insertion is labeled with red. Na+/K+ ratio contents in roots of TS-21 wild type and slhak20 mutants. Data are shown as means ± SD (n = 4). Na+ contents in roots of TS-21 wild type and slhak20 mutants. Data are shown as means ± SD (n = 4). K+ contents in roots of TS-21 wild type and slhak20 mutants. Data are shown as means ± SD (n = 4). Na+ contents in the xylem sap of TS-21 and slhak20 mutants after 50 mM NaCl treatment for the indicated days. Data represent means ± SD (n = 4). K+ contents in the xylem sap of TS-21 and slhak20 mutants after 50 mM NaCl treatment for the indicated days. Data represent means ± SD (n = 4). G Shoot height of TS-21, slhak20-1, and slhak20-2. Plant height of 24-day-old TS-21, slhak20-1, and slhak20-2 were measured after growing under normal conditions for 14 days, followed by salt treatment for 14 days, and recovery for 10 days. Values are means ± SD (n = 10 plants of each genotype). H Salt resistance of TS-21 wild type and slhak20 mutants. TS-21, slhak20-1, and slhak20-2 grown under normal growth conditions for 24 days, followed by a treatment with 200 mM NaCl for 14 days, and then recovered for 10 days. Control plants grown under normal growth conditions for 38 days. Scale bars, 5 cm.
CircRNA abundance is brain region-dependent and PD-modified (A) mRNA processing either involves unidirectional splicing, creating linear mRNA transcripts or back-splicing generating circRNAs.Figure 2(A) mRNA processing either involves unidirectional splicing, creating linear mRNA transcripts or back-splicing generating circRNAs. (B,C) Venn diagram (not to scale) showing brain region- and disease-characteristic circRNA abundance patterns in analyzed brain regions and disease state. (D) Heatmap of circRNA abundance, indicating significant tissue-based SN clustering, PD samples indicated in red. (E,F) Pie chart demonstrating brain-specific expression of mRNAs (E) in comparison to circRNAs (F). (G) Venn diagram demonstrating predicted cell-type characteristics of circRNA host genes in the SN.
Figure 7A Representative tile scan 3D images with α-SMA, Isolectin, FABP4 and Emcn in the pancreas from 17-weeks-old Gja1i∆EC mutant and control mice. Insets show higher magnifications of regions.G Representative 3D images of ESM-1 and Caveolin-1 in pancreatic islets from Gja1i∆EC and control mice. Insets (* and #) show higher magnifications with surface rendering for ESM-1 and Caveolin-1 immunostaining. Combo plot with the quantification of ESM-1+ cell numbers per islet (f.u). (n=5), P-value, and two-tailed unpaired t-test. The scale bars for the 3D images are 50 µm, and 5 µm for the higher magnifications with single-cell resolution images. The dashed lines in panel C, E, G, H and I represent the outlines of pancreatic islets. Two-tailed Student's t-test were performed for the statistical analysis. **: P < 0.01; ****: P < 0.0001. Nuclei: TO-PRO-3 as indicated. Is: pancreatic islet. f.u: fluorescence unit. H 3D images with CXCR4 and Emcn immunostaining in Gja1i∆EC and control pancreas. The scale bars are 50 µm.
Second site mutation in MAKR5 partially suppresses brx root phenotypes.Figure 2(A) Root phenotypes of 7-day-old seedlings of indicated genotypes. (B) Primary root length of 7-day-old seedlings. (C) Root meristem size in 5-day-old seedlings. (D) Root meristem phenotype of 5-day-old seedlings, confocal microscopy, propidium iodide-stained (red). (E) Auxin activity as visualized by the constitutively expressed inverse DII-NLS-VENUS marker (green) in root meristems of 5-day-old seedlings. (F) Gap cell frequency in brxsingle or brxmakr5 double mutants (note: gap cells were absent from Col-0 wild type or makr5single mutants). (G) Illustration of gap cells (arrowheads) in developing protophloem sieve element strands (asterisks) of brxsingle or brxmakr5 double mutants.
Multiple-perturbation phosphoproteomic experiment using R2-P2 and DIA-MS Scheme of the experimental setup.Figure 4Scheme of the experimental setup. Σ1278b MATa yeast cultures were grown and treated in biological triplicates with the indicated stimuli in a three-point time course. Proteins were purified and digested using R2-P2 with Fe3+-IMAC or Ti4+-IMAC. The spectrum library was generated by DDA measurement of pooled time points of Fe3+-IMAC and Ti4+-IMAC enriched samples. The chromatogram library was generated by measuring a pooled sample from Fe3+-IMAC enrichment of all conditions and time points in seven staggered narrow-window DIA-MS runs. The Fe3+-IMAC fractions of each sample were measured in a single injection wide window DIA-MS experiment. The time used for sample preparation and measurement is indicated on the top and numbers of identified phosphopeptides on the right. Unique phosphopeptides present in at least two (out of three) replicates identified in the DIA-MS experiment.   Boxplot depicting distributions of Pearson correlations for biological replicates and different treatments for different time points. P-values are calculated by Wilcoxon rank-sum test.   Significantly regulated phosphopeptide isoforms for the different stimulation and time points compared to the corresponding untreated control as determined by a two sample t-test (permutation-based FDR < 0.05, and fold change > 1.5). Only phosphopeptide isoforms with all phosphosites localized were considered.
a) Western blot analysis of GEMIN4 in fractions from a discontinuous gradient of Histodenz (15%, 20%, 24%, 26%) described to enrich autophagosomes (light AV) and autophagolysosomes (heavy AV).figf3(a) Western blot analysis of GEMIN4 in fractions from a discontinuous gradient of Histodenz (15%, 20%, 24%, 26%) described to enrich autophagosomes (light AV) and autophagolysosomes (heavy AV). HEK293T cells were treated with CQ (20 μM, 16 h). Fractions enriched in multivesicular body (MVB) and lysosomal markers are indicated. (b) Western blot of GEMIN4 or loading control (TUBA) in HeLa cells treated with control siRNA or siRNA targeting ATG5, ATG6 or ATG7. (c) Western blot analysis of GEMIN4 or control (TUBA) in HeLa cells cultured in DMEM 10% FBS (Serum), Hank's buffered salt solution (Starve) for 2 h, or treated for 16 h with control (DMSO), BAF (200 nM), RAP (20 nM), RAP+BAF, or PP242 (100 nM). (d) Top, western blot of GEMIN4 immunoprecipitates (IP) from HeLa cells. Bottom, western blot analysis of GEMIN4 in immunoprecipitates of anti-NDP52, -p62 or control antibody from HeLa cells. (e) Western blot of DICER immunoprecipitates from HeLa cells. (f) Western blot of AGO2 immunoprecipitates from HeLa cells. (g) Western blot analysis of GEMIN4, compared with loading controls (DCP1A, CLN3) in HeLa cells treated with control siRNA or siRNA targeting GEMIN3 and GEMIN4. (h) Western blot analysis of HeLa cells treated with indicated combinations of control siRNA or siRNA targeting GEMIN3, GEMIN4 and ATG5. Western blot analysis of CLN3 and DCP1A are used to demonstrate equal loading of wells. (i) Western blot analysis with anti-UB monoclonal antibody (mono- and polyubiquitylated proteins, FK2 clone) in Flag immunoprecipitates performed under stringent denaturing conditions. Flag-AGO2 was induced for 24 h with tetracycline in HEK293T cells with Tet-inducible Flag-AGO2 stably integrated that had been treated 24 h before with the indicated siRNAs. (j) Western blot analysis of HeLa cells treated with control siRNA or siRNA targeting ATG5 for 24 h. TUBA, α-tubulin loading control. (k) Top, RT-qPCR analysis of miR-16 or let-7a levels in AGO2 immunoprecipitates from DMSO- and BAF-treated HeLa cells. Error bars represent s.e.m. Bottom, a representative western blot of AGO2 immunoprecipitates used in one replicate of these experiments. Uncropped images of blots are shown in Supplementary Fig. S3.
| Comprehensive proteomic and transcriptomic analysis of 29 human tissues from healthy donors.Figure 1Body map of analysed tissues. Number of genes detected on protein and mRNA level in each tissue. The colouring the bars indicates the fractions of transcripts and proteins that are expressed everywhere or enriched in certain tissues. The full classification is provided in the text. Abundance distribution of all transcripts detected in all tissues (grey); the fraction of detected proteins is shown in blue and the fraction of transcripts for which no protein was detected is shown in orange. Relative distribution and absolute numbers of transcripts and proteins in selected functional classes across the expression categories shown in panel B.
Figure 3ELISA of IL-1β secretion by WT or CYLD KO BMDMs that were either untreated (-) or LPS-primed and then treated with the indicated NLRP3 inducers. Immunoblot analysis of IL-1β and active caspase-1 p20 in cell supernatants (Sup) and the indicated proteins in cell extracts (Cell ext) from WT and CYLD KO BMDMs, stimulated as in (A). BMDMs from WT and CYLD KO mice were pretreated with Caspase-1 inhibitor, Z-YVAD-FMK, for 30 min, primed with LPS, and then stimulated with Nigericin for 30 min. ASC specks were assessed by Immunofluorescence with antibody against ASC and the percentage of ASC speck-positive cells was quantified. LDH release from WT and Cyld KO BMDMs primed with LPS and stimulated as indicated. Immunoblotting analysis of active caspase-1 p20 and IL-1β in cell supernatants (Sup) and the indicated proteins in cell extracts (Cell ext) of WT iBMDMs and Spata2 KO iBMDMs reconstituted with WT Spata2 or a Spata2 mutant (F108A) defective in CYLD binding that were primed with LPS and stimulated with Nigericin for 1 h.
Effects of Atg16L1 mutations on autophagy against Salmonella.Figure 6.(A) Schematic diagram of Atg16L1 and various mutants. The magenta box indicates 239-242A and the cyan box indicates 194-195A. (B-E) Atg16L1-Δ/Δ MEFs stably expressing the indicated constructs were infected with Salmonella (MOI = 10) for 1 h and then analyzed by immunocytochemistry for LC3 (B) or Atg16L1 (D). Bar, 5 µm. The percentage of LC3- or Atg16L1-positive Salmonella per Ub-positive Salmonella was enumerated by fluorescence microscopy (C and E). At least 50 bacteria were counted. The average ± SD is shown for three independent experiments. (F) Atg16L1-Δ/Δ MEFs stably expressing the indicated constructs were cultured in growth medium (Fed) or EBSS (Starved) for 1 h and then subjected to immunocytochemistry using an anti-LC3 antibody. The number of LC3 puncta in each cell was counted for more than 50 cells. The average ± SD is shown for three independent experiments.
AOX expression prolongs the survival of cIII deficient Bcs1lp.Figure 1A. Schematic presentation of the multiorgan manifestations, described in this study or previously, in homozygous Bcs1lc.A232G (GRAC) mice, and the time points of the investigations performed in this study B. Survival curves of Bcs1l homozygotes without (GRAC) and with (GROX) alternative oxidase (AOX) expression (n=18-21/group). The median survival of GRAC mice was 210 days and of GROX mice 589 days with no gender difference Statistics: The survival data was analyzed using Log-rank (Mantel-Cox) test (p<0.0001) C. Weight of mice from P50 to P200 (n>10/group) and at P600 (n=4-6/group). D. Blood lactate to glucose ratio at P200 (n=8/group) Bar graphs represent mean±SD The dat were analyzed using Kruskal-Wallis and Mann-Whitney U tests for selected comparison. Significant differences between groups (p value) are indicated on graphs Dual energy X-ray absorptiometry (DEXA) analysis (n=10/group) of (E) lean mass, (F) fat mas at P98 Bar graphs represent mean±SD. The dat were analyzed using Kruskal-Wallis and Mann-Whitney U tests for selected comparison. Significant differences between groups (p value) are indicated on graphs Dual energy X-ray absorptiometry (DEXA) analysis (n=10/group) o (G) bone mineral density at P98 Bar graphs represent mean±SD. The dat were analyzed using Kruskal-Wallis and Mann-Whitney U tests for selected comparison. Significant differences between groups (p value) are indicated on graphs Indirect calorimetric measurement (n=10/group) of (H) heat productio at P77 Bar graphs represent mean±SD The dat were analyzed using Kruskal-Wallis and Mann-Whitney U tests for selected comparison. Significant differences between groups (p value) are indicated on graphs Indirect calorimetric measurement (n=10/group) o (I) respiratory exchange ratio at P77 Bar graphs represent mean±SD The dat were analyzed using Kruskal-Wallis and Mann-Whitney U tests for selected comparison. Significant differences between groups (p value) are indicated on graphs
Figure 7High time resolution imaging of the membrane marker CD4::mIFP expressed under btl-gal4. (A) Example of a large CD4 vesicle (arrowhead) rapidly moving towards the tip of the terminal cell. (B-D) Analysis of 28 CD4 vesicles in five terminal cells. (B) Trajectories of CD4 vesicles with their original position mapped to the origin of the plot. (C) Rose diagram of the trajectories shown in (B). (D) Speed of CD4 vesicles compared to speed of tube growth measured over 25 min. Data are plotted as mean +/-SD. (E-H) Terminal cells expressing the membrane reporter CD4::mIFP together with markers for vesicles of the endocytic pathway. (E) Rab5::YFP, red arrowheads point to a Rab5-positive vesicle; the white arrowhead shows the same vesicle once it lost the Rab5 signal. (F) FYVE::GFP, a PI3P reporter and Spin::mRFP , a lysosomal marker. Black arrowheads point to small FYVE::GFP vesicles and the squared area shows large ones at the tip. (G) Par6::mCherry and endogenously labelled Rab7::YFP. (H) Percentage of CD4::mIFP vesicles that carry the indicated markers. Data are plotted as mean +/-SD. For each marker we analyzed at least 20 minutes of cell growth with 10-20 timepoints. Number of cells analyzed: Par3, n=2; Rab5, n=3; FYVE::GFP, n=4; FYVE::GFP-Spin::RFP (which includes single- and double-positive vesicles), n=3; dLamp1, n=2; Rab7, n=5.
RNA depletion does not affect TAD Boundaries Interaction heatmaps at 40kb resolution zooming in chr21:19.Figure 3Interaction heatmaps at 40kb resolution zooming in chr21:19.5-33Mb and showing the TAD structures in aXL and bXL datasets. The aXL and bXL RNase A / control log2 ratios are shown below the heatmaps. Boxplot showing the TAD boundary scores for the aXL and bXL datasets. p-value: Wilcoxon rank-sum test. (*** p < 0.05). The horizontal bands in the boxplots represent the median, the error bars represent +/- 1.5 x IQR values, and the outer shapes represent the density of the data points. The figures represent data generated from the pooled Hi-C datasets with 2 biological replicates. C-D) Mean interactions frequencies +/- 1Mb centered on all TAD boundaries for each dataset at 40kb resolution, as well as the log2 ratios of aXL (left panel) or bXL (right panel) samples. The TAD structures are not altered upon RNase A treatment in samples treated with RNase after crosslinking.
Fig.Figure 5A Sequence chromatograms of 3'UTR in all three passaged viruses (P50-A, P50-B and P50-C). B RT-PCR analysis of different passages of viruses using the primer pair spanning the region from C-terminus of NS5 to CS1 region of 3'UTR. The primer sequences were described in Materials and methods. C-D Plaque morphologies (C) and growth kinetics (D) Growth kinetics from P10, P30 and P50 using plaque assay. Three independent assays were performed and the data of one representative experiment was presented. Values indicate mean ± sd from two replicates of the experiment. Data information: For statistical analysis, two-way ANOVA analysis were used **p<0.01, *** p<0.001,****p< 0.0001, ns represents not significant. Virulence and efficacy of the passaged WNV-poly(A) (WNV-poly(A)-P50) in mice (n=5). (E) Survival of mice infected with 107 PFU of WNV-poly(A)-P50. Data information: For statistical analysis, one-way ANOVA analysis and Log-rank test were used **p<0.01, *** p<0.001,****p< 0.0001, ns represents not significant. Virulence and efficacy of the passaged WNV-poly(A) (WNV-poly(A)-P50) in mice (n=5). (F) Antibody titers of mice infected with WNV-poly(A)-P50 at different days post immunization. (For the values represent mean ± sd in each group (n=5) and the horizontal dotted line represents the limit of detection). Data information: For statistical analysis, Mann-Whitney test were used **p<0.01, *** p<0.001,****p< 0.0001, ns represents not significant. Virulence and efficacy of the passaged WNV-poly(A) (WNV-poly(A)-P50) in mice (n=5). (G) Neutralizing antibody titers of mice infected with WNV-poly(A)-P50 at different days post immunization. (For G, the values represent mean ± sd in each group (n=5) and the horizontal dotted line represents the limit of detection). Data information: For statistical analysis, Mann-Whitney test were used **p<0.01, *** p<0.001,****p< 0.0001, ns represents not significant. (H) Viremia of mice after WT WNV challenge. (The values represent mean ± sd in each group (n=5), each symbol represents a mouse). Data information: For statistical analysis, one-way ANOVA analysis and Log-rank test were used **p<0.01, *** p<0.001,****p< 0.0001, ns represents not significant. (I) Survival of mice after WT WNV challenge. Data information: For statistical analysis, one-way ANOVA analysis and Log-rank test were used **p<0.01, *** p<0.001,****p< 0.0001, ns represents not significant.
90 and 1-277 regions of M45 confer protection against TNF-induced necroptosis in human cells.Figure 1Sequence alignment of RHIMs from M45 and human and murine RIPK1, RIPK3 and ZBP1 Schematic representation of M45, RIPK1 and RIPK3 proteins. Grey regions indicate RHIM-encompassing fragments used in this study. Black bar represents 19-residue RHIM. RNR-like: inactive ribonucleotide reductase domain; DD: death domain; Z: double-stranded RNA-specific adenosine deaminase (DRADA) repeat Electron micrograph of fibrils formed by a synthetic peptide encompassing the M45 RHIM. Representative of three samples examined. Scale bar 100 nm HT29 cell survival 18h after treatment with TNF+BV-6+z-VAD-fmk to induce necroptosis. Cells expressed lentivirus constructs encoding 1-90 or 1-277 fragments of M45 and were cultured +/- the addition of coumermycin A1 to induce dimerisation. N=2 for M45-90 gyrase +/- coumermycin A1 and 4 for M45-277 gyrase and for empty vector, +/- coumermycin A1
promotes the autophagic degradation of MAVS aggregates (A) Immunoblot showing the degradation of MAVS in HEK293T cells subjected to VSV infection (MOI = 1.Figure 5(A) Immunoblot showing the degradation of MAVS in HEK293T cells subjected to VSV infection (MOI = 1.0) for the indicated times. α-Tubulin was used as a loading control. Cell-based studies were performed independently at least three times with comparable results. (B) Immunoblot showing the degradation of MAVS in HEK293T cells infected with VSV (MOI = 1.0) for the indicated times in the presence or absence of NH4Cl (3 mM). α-Tubulin was used as a loading control. Cell-based studies were performed independently at least three times with comparable results. (C) Immunoblot showing the levels of the MAVS protein in HEK293T cells expressing Flag-MAVS or Flag-mMAVS together with Myc-RNF34. α-Tubulin was used as a loading control. Cell-based studies were performed independently at least three times with comparable results. (D) Immunoblot showing the levels of the MAVS protein in HEK293T cells expressing Flag-MAVS or its 4KR mutant together with increasing amounts of Myc-RNF34. α-Tubulin was used as a loading control. Cell-based studies were performed independently at least three times with comparable results. (E) Immunoblot showing the levels of the MAVS protein in HEK293T cells expressing Flag-MAVS with increasing amounts of HA-RNF34 or its H342A mutant. α-Tubulin was used as a loading control. Cell-based studies were performed independently at least three times with comparable results. (F) SDD-AGE analysis of MAVS aggregates in HEK293T cells expressing Flag-MAVS together with WT Myc-RNF34 or its ligase-dead mutant and cultured in the presence or absence of MG132 (10 μM), 3-MA (1 mM) or NH4Cl (3 mM). SDS-PAGE immunoblotting was used as a loading control. Cell-based studies were performed independently at least three times with comparable results. (G) SDD-AGE analysis of MAVS aggregates in siCtrl- or siRNF34-1-transfected cells using an anti-MAVS antibody. SDS-PAGE immunoblotting was used as a loading control. Cell-based studies were performed independently at least three times with comparable results. (H) Immunoblot showing the levels of the MAVS protein in siCtrl- or siRNF34-1-transfected PBMCs. α-Tubulin was used as a loading control. Cell-based studies were performed independently at least three times with comparable results. (I) SDD-AGE analysis of MAVS aggregates in THP-1 cells expressing Flag-MAVS or the K311R mutant together with Myc-RNF34 or Myc-TRIM31. SDS-PAGE immunoblotting was used as a loading control. Cell-based studies were performed independently at least three times with comparable results.
Figure 4(A) Principal component analysis of WT CD4+ naïve T cell (gray), WT Treg cells (blue), and Tet2/3 DKO Treg cells (red). The percent of variation explained by principle components 1 (PC1) and 2 (PC2) are shown in parenthesis. (B) Scatter plot of ATAC-seq data for WT Treg vs Tet2/3 DKO Treg cells. Differentially accessible regions (DARs) are shown in color and the total number of DARs (N) is shown on the graph. (C) K-means clustering of ATAC-seq data for DARs between WT Tregs and Tet2/3 DKO Tregs. ATAC-seq data for CD4+ naïve T cells is from Yoshida et al, 2019. The number of regions in each cluster is denoted. (D) Heatmap of transcription factor binding motifs in each K-means cluster. The significance of enrichment (-log10(FDR)) is denoted by color scale. Transcription factor families of particular interest are highlighted on top of the heatmap. (E) Genome browser view of chromatin accessibilities at Hopx and Il10 loci for CD4+ naïve T cells, WT Treg cells and Tet2/3 DKO Treg cells. DhmRs, DM-CpGs and DARs (shaded) are shown on top of the tracks. Conservation track (Cons) is shown at the bottom. (F) Expression levels of Hopx and Il10 in CD4+ naïve T cells, WT Treg cells and Tet2/3 DKO Treg cells. Error bars show mean ± S.D. from two biological replicate samples.
MAP1S phosphorylation by CDKL5 impairs its microtubule binding.Figure 6 A Schematic representation of MAP1S protein domains. Phosphorylation sites S786 and S812 (green Ps) are on the microtubule binding domain of MAP1S LC. Scissors indicate proteolytic cleavage  B COS-7 cells expressing HA-MAP1S LC, Flag-CDKL5 WT / kinase dead (KD) is shown with endogenous α-tubulin staining. MAP1S does not localize to microtubules when phosphorylated by CDKL5. Scale bar is 20 μm  C, D The percentage of cells where α-tubulin and MAP1S LC are colocalised (C) and the quantification of MAP1S LC and α-tubulin colocalisation by Pearson correlation coefficient (D). Tukey's multiple comparison: n = 22-24 cells per condition  E, F Taxol-stabilised microtubule co-sedimentation assay shows direct binding of MAP1S LC to microtubules is reduced upon prior phosphorylation by CDKL5. Quantification of soluble MAP1S in the supernatant (s/n) fraction is normalized to the MT-bound pellet fraction. 3:1 MT:MAP1S LC ratio used here. Tukey's multiple comparison: n = 3 technical repeats  G HA-MAP1S LC is overexpressed and stained with anti-HA antibody in cultured cortical neurons (red). Numerous loops at the dendrites' tips are observed (arrowheads). Scale bar is 5 μm