EMBO/sd-character-level-ner huggingface dataset:
Figure 5(A) SFA/UFA ratios of WT and indicated mutant strains overexpressing SCT1 (pSCT1) vs. control (pEmpty). Data is presented as mean ± SD (n≥3). * p < 0.05, ** p < 0.01, **** p < 0.0001, n.s. not significant, unpaired two-tailed t-test of the indicated bar compared to WT pSCT1. (B) Growth of WT and indicated mutant strains overexpressing SCT1 (pSCT1) vs. pEmpty. Serial dilutions (10-1 - 10-4) were spotted on SGal + 0.05% glucose and incubated for 3 days at 30°C. PS decarboxylase activity in wild-type (WT, BY4741) and the indicated mutant strains transformed with pEmpty, expressed as (C) percentage of the lipid-incorporated 13C15N-label recovered in PE, The differences in molecular species profiles of *PS and *PE between the BY4741 WT shown here and those obtained by 2H3-serine labeling (Figure 4A) are attributed to the different culture media, lipid extraction procedures and mass spectrometry techniques used. PS decarboxylase activity in wild-type (WT, BY4741) and the indicated mutant strains transformed with pEmpty, expressed , and (D) molecular species composition of 13C315N-labeled PS (*PS) and 13C215N-labeled PE (*PE) after 20 min incubation with 13C315N-serine. The differences in molecular species profiles of *PS and *PE between the BY4741 WT shown here and those obtained by 2H3-serine labeling (Figure 4A) are attributed to the different culture media, lipid extraction procedures and mass spectrometry techniques used. (E) The corresponding molecular signatures of PSD activity represented by the label distribution per molecular species between *PS and *PE as percentages of the total amount of label incorporated in the molecular species indicated.
ECE2 KO affects neural progenitor polarity and actin and microtubule cytoskeleton in COs.Figure 4Quantification of actin levels from Westernblot of whole CO lysate reveals a reduction in ECE2 KO COs (n=4 independent batches of CTRL and ECE2 KO COs with 3 pooled COs each sample; *P = 0.021 in Kruskal-Wallis One Way Analysis of Variance on Ranks and Dunn's Pairwise Comparison). Fractionation of G- and F-actin from CTRL and ECE2 KO COs and analysis via Westernblot additionally reveals an increase in G- at the expense F-actin upon ECE2 KO, suggesting a reduction in F-actin belt integrity (n=4 independent batches of CTRL and ECE2 KO COs with 3 pooled COs each; *P = 0.021 in Kruskal-Wallis One Way Analysis of Variance on Ranks and Dunn's Pairwise Comparison). The thickness of the apical F-actin belt is increased in ECE2 KO COs. C, Example images of apical F-actin belt in 60d old CTRL and ECE2 KO COs with F-actin labeled by AlexaFluor594-conjugated Phalloidin (Scalebar=100 µm). D, Quantification of the thickness of apical F-actin belt in Fiji [86] by measuring the area of F-actin and dividing by the length of apical surface reveals significant increase in ECE2 KO COs (Box plots: mean=red line, median=black line, box represents 25th and 75th percentiles, whiskers extend to 10th and 90th percentiles, all outliers are shown; n=number of analysed ventricles in 2 batches; Kruskal-Wallis One Way ANOVA on Ranks and Dunn's Pariwise Multiple Comparison: ***P < 0.001). IHC for ARL13B and F-ACTIN in 60d old CTRL and ECE2 KO COs shows disruption of honeycomb-like structure of the apical adherens belt and of apico-basal polarity in terms of apically localised primary cilium in ECE2 KO COs (Scalebar = 10µm). The microtubule cytoskeleton is changed in COs upon ECE2 KO. Example images of ac-tub IHC in CTRL vs. ECE2 KO COs (Scalebar = 100 µm). The microtubule cytoskeleton is changed in COs upon ECE2 KO. Quantification of ac-tub in ventricles of ECE2 KO COs by measurement of the mean grey value in Fiji shows significant reduction upon ECE2 KO (n=number of analysed ventricles; *P = 0.019 in One Way ANOVA and Tukey Pairwise Multiple Comparison). Apico-basal polarity is impaired upon ECE2 inhibition and KO as visible in example images of ARL13B IHC in CTRL vs. KO COs (H; Scalebar = 100 µm). Quantification of germinal zones for normally high ("1") vs. reduced ("0") apical ARL13B reveals a reduction in the proportion of normal ventricles in the absence of ECE2 (n=number of analysed ventricles from 2 batches; ****P < 0.0001 in exact binomial test). Analysis of CTRL and ECE2 KO COs 1 dpe with GAP43-GFP reveals RG that delaminated (white arrowheads) and/or lost their bipolar morphology (red arrowheads) in the KO COs (Scalebar = 25 µm).
Endogenous presentation of a peptide derived from cytosolic NeoR on HLA‐DP3.Figure 1(A) The T cell clone 20-4/A4 recognizes an epitope derived from the NeoR protein in the context of HLA‐DP3. MHC class II expression on RCC1.24‐NeoR cells following IFN‐γ treatment confers recognition by 20-4/A4. This recognition is almost completely blocked by the HLA‐DP‐specific antibody B7/21. EBV‐B cells transfected with pINCO‐NeoR are able to present endogenous NeoR on MHC class II. As exemplified by the cell lines EBV‐B1.11 (HLA‐DP3) and EBV‐B1.25 (HLA‐DP4), recognition by 20-4/A4 is dependent on HLA‐DP3 expression. Bacterially expressed NeoR protein and a synthetic peptide encompassing amino acids NeoR216-229 served as controls. (B) Cross-presentation experiments to exclude release of NeoR and re‐uptake as exogenous protein. EBV‐B1.25 cells transfected with the neoR expression plasmid pINCO‐NeoR, were co‐cultured for 24 h with the same number of untransfected EBV‐B1.11 cells, or vice versa. Then, T cells were added and co‐cultured with the cells for additional 24 h. To test for NeoR release by neoR‐transfected EBV‐B1.11 cells, total supernatant (200 μl) of 1×105 EBV‐B1.11 cells was harvested 48 h post transfection by centrifugation, and was used as culture media for 1×105 untransfected EBV‐B1.11 cells. After 48 h, 1×105 20-4/A4 T cells were added and GM‐CSF concentration determined after additional 24 h.
Enhanced sensitivity of atg5, atg7, and nbr1 mutants to heat stress.Figure 6(A) Electrolyte leakage. (B) Fv/Fm images. (C) Average values for the Fv/Fm images. Five weeks-old Arabidopsis Col-0 wild type (WT), atg5, atg7 and nbr1 mutant plants were placed in 22°C and 45°C growth chambers. Fully expanded leaves were sampled after 10 hours in the indicated temperatures and immediately measured for electrolyte leakage and Fv/Fm. Error bars in (A) and (C) indicate SE (n = 5). According to Duncan's multiple range test (P = 0.05), means of EL or Fv/Fm do not differ significantly if they are indicated with the same letter. The experiments were repeated twice with similar results.
Figure 1Schematic diagram of the PAX6 isoforms in human. Western blot detection of PAX6 isoforms in human fetal forebrain and retina. The antibody used targets the C terminus of PAX6 and can detect all the 3 isoforms at different molecular weight. The green arrow indicates the PAX6A&B isoforms. The red arrow indicates the PAX6D isoform. #1, #2 are #3 are from ~8-week-old human embryos. #4 is from ~12-week-old human fetus. qPCR quantification of overall PAX6 gene expression during human forebrain and retinal differentiation (n=3 biological replicates for each group). qPCR quantification of PAX6A&B isoform expression during human forebrain and retinal differentiation (n=3 biological replicates for each group). qPCR of PAX6D isoform expression during human forebrain and retinal differentiation (n=3 biological replicates for each group). Western blot detection of PAX6 isoforms in cells during forebrain and retina differentiation from hESCs. D10 indicates day 10 of differentiation. The green arrow indicates the PAX6A&B isoforms. The red arrow indicates the PAX6D isoform.
Clathrin‐mediated endocytosis is required for necrotic cell death in C.Figure 1(A) Number of cell corpses at the L1 stage of development per 100 animals carrying the neurotoxic mec‐4(d) or deg‐3(d) alleles in SNT‐1‐deficient mutants. For mec‐4(d) mutant animals, bars denote touch receptor neuron corpses. For deg‐3(d)mutants, bars denote inner labial neuron 1 (L1) sensory neuron and PVC interneuron corpses. (B) Expression of a full‐length MEC‐4::GFP reporter fusion under the control of the mec‐4 promoter in touch receptor neurons of wild‐type, snt‐1 and unc‐57 mutant animals. Representative photos of touch receptor neuron cell bodies are shown. Bar denotes 6 μm. The quantification (%) of GFP signal intensity from the animals examined is graphed below (n=100 neurons per assay; P0.05 compared with wild type; t‐test). (C) Percentage of SNT‐1‐deficient animals that survive near‐lethal treatment with sodium azide. This chemical inhibits the activity of the respiratory electron transport complex IV (cytochrome C oxidase) and simulates hypoxia. (D) Number of touch receptor neuron corpses, at the L1 stage of development, per 100 animals carrying the neurotoxic mec-4(d) or deg‐3(d) alleles in UNC‐57‐deficient animals. (E) Percentage of UNC‐57‐deficient animals that survive after the hypoxia‐inducing treatment with sodium azide. (F) Number of touch receptor neuron corpses, at the L1 stage of development, per 100 animals carrying the neurotoxic mec‐4(d) or deg‐3(d) alleles together with lesions in genes encoding key proteins that participate in all four steps of clathrin‐mediated endocytosis. UNC‐11 and DPY‐23 are adaptor proteins required for the formation of clathrin‐coated pits, DYN‐1 is a GTPase necessary for fission of clathrin‐coated vesicles and UNC‐26 participates in the uncoating of vesicles. Error bars denote s.e.m. values (n>250 for all populations examined; P0.001, compared with wild‐type animals, unpaired t‐test).
Figure 1Structure of quiescent SecYEG of Thermus thermophilus in the lipid cubic phase (PDB ID: 5AWW). TMHs 2b, 3, 7, and 8 of the lateral gate, as well as the proximate loop 6/7 involved in ribosome binding are indicated. The non-essential SecG subunit is omitted for clarity. Model of the SecY lateral gate opening upon inserting a nascent chain (red) in the lipid bilayer. The color-coding of SecYE TMHs is as in panel A. In the presence of the completely inserted and folded nascent chain, TMHs 2b and 3 of the N-terminal domain of SecY are displaced (arrows) thus opening a broad passage for the nascent TMH towards the lipid moiety. SDS-PAGE of SecYEG-ND sample after size-exclusion chromatography. Asterisks indicate translocon-enriched fractions used for forming the RNC FtsQ:SecYEG-ND complex. Lipid-loaded "empty" nanodiscs elute at larger volumes and so can be separated. Schematic drawing of a SecYEG-ND particle. Lateral dimensions of the nanodisc should be appropriate to accommodate a single SecYEG with surroundings lipids, thus mimicking the naturally occurring environment.
Figure 1Illustrated schematic representation of our single-molecule assay. Cas9 assembled with tracrRNA and Cy5-labelled crRNA is immobilized on the surface via biotin-streptavidin links (left). After addition of Cy3-labelled DNA substrate interaction between Cas9 and DNA brings the dyes together resulting in FRET signal (right). EmCCD images show Cas9:RNA immobilized on the surface with green and red laser illumination (left). No fluorescence is seen when illuminated with green laser. Immobilized Cas9 can be seen when illuminated with red laser. After addition of Cy3-labeled DNA spots appear on the EmCCD in the green channel when illuminated with green laser (right). Size of EmCCD images is 25µm x 25µm. Example time trace when no binding event occurs (green laser excitation). EmCCD image shows signal from green and red channels before the addition of Cy3-labelled DNA when illuminated with green laser. Size of EmCCD image is 25µm x 50 µm.   Example time trace showing a binding event (green laser excitation). EmCCD image shows signal from green and red channels after the addition of Cy3-labeled DNA when illuminated with green laser. Size of EmCCD image is 25µm x 50 µm.
Figure 3A Representative western blot analysis of recognized autophagy signaling molecules (pJNK, JNK, pAKT, and AKT, pmTOR, and mTOR) in HC (#1-3) and DM (#1-8) patient platelets. Quantification analysis of autophagy upstream signaling molecules on HC (#1-5) and DM (#1-18) individuals. (pAKT/AKT; p=0.0747, pJNK/JNK; *p=0.001, pmTOR/mTOR; p=0.3356 vs. HC). GAPDH was used as the loading control. B Representative western blot analysis using HC and HG platelets with or without H2O2. And assessing for LC3I/II. GAPDH was used as the loading control. C Platelet suspensions were incubated in HG for 2 hours and with H2O2 (1mM). ROS levels were evaluated using a detection kit (Enzo Life Science) for 60 mins at 37 ºC. D Western blot analysis of p53, JNK and LC3I/II in HC platelets treated with H2O2 (1mM for 1 hour) alone or with NAC (100 μM for 30 min). GAPDH was used as the loading control. E Confocal microscopy was used to corroborate the Western analysis using triple staining for Mitotracker, CoxIV, and LC3 in HC or H2O2. The insets highlight the association of low membrane potential with high levels of LC3 and increased colocalization of Cox IV with LC3 in H2O2-treated platelets. F HC platelets were pretreated with H2O2 to induce autophagy and then treated with or without SP600125 (JNK inhibitor) at 3 concentrations (1, 5 and 10 µM) to assess for inhibition of autophagy. This Western blot is representative of three independent experiments. Quantification of the three independent experiments is provided at right. (pJNK/JNK in H2O2/SP600125; **p=0.0286 vs. H2O2 group, LC3II in H2O2; *p=0.0241 vs. HC group, LC3II in H2O2/SP600125; **p=0.0044 vs. H2O2 group, n=3 for each group). G Triple staining of CoxIV, LC3, and LAMP1 in HC and H2O2 with/without SP600125-treated platelets using confocal microscopy. Arrow indicates localization of LC3 and LAMP1. Graph indicates colocalization between LC3, LAMP1 and CoxIV signal. The y-axis indicates fold change of colocalization between LC3 and LAMP1 in the mitochondria area. Signal intensity of each group was converted to fold and compared with HC values (H2O2;*p=0.023 vs. HC, H2O2/SP600125;*p=0.04 vs. H2O2 group).
a–g, Proteostasis analysis in TIS control versus senescence-incapable lymphoma cells in vitro, as in Fig.f3Proteostasis analysis in TIS control versus senescence-incapable lymphoma cells in vitro, as in Fig. 2, or versus SASP-attenuated TISlymphoma cells. a, Global protein synthesis rate, measured by incorporation of green-fluorescent methionine. Mean fluorescent intensities reflect newly synthesized protein within 2 h (DAPI used as a counterstain). Data represent means ± s.d. (n = 5 each). b, Rate of global protein synthesis (measured as in a) in ADR-senescent relative to untreated (ut) control;Bcl2 lymphoma cells; either empty vector-infected cells or cells stably expressing the NF-kB super-repressor IkBaDN(SR, ref. 18). Results represent means ± s.d. (n = 5 each). c, Relative intracellular glucose concentrations in cells as in b. d, Relative OCR in cells as in b. e, Relative intracellular ATP concentrations in cells as in b f, Expression of the transcription factors Xbp1 (by RT-PCR (PCR with reverse transcription; top); note that the lower band reflects the UPR-related splice form), ATF4, CHOP, phospho-Jun N-terminal kinase (JNK-P-Thr183/Thr185) and total JNK(by immunoblot analysis, with a-tubulin as a loading control; bottom) in lymphoma cells as indicated (n = 3 each, gels were cropped to ease presentation; full-length blots are provided in Supplementary Fig. 16). g, Global protein ubiquitination, expression of p62/SQSTM1 (whose transcript levels were also selectively induced in TIS cells by approximately 4.8-fold (data not shown)) and V-ATPase subunit A1, and conversion of MAP1-LC3-I to MAP1-LC3-II by immunoblot analysis, demonstrating autophagosome formation in lymphoma cells as in f (α-tubulin was used as a loading control, gels were cropped to ease presentation; full-length blots are provided in Supplementary Fig. 16). *P<0.05.
Figure 2A Ubiquitylation assay using H3N-GST. The components included in the assay and the ubiquitylated H3N-GST species are indicated at right. B MS/MS spectrum of the histone H3 peptide corresponding to residues 10-17. The observed y and b ions and fragment map are shown. C Ubiquitylation assay using biotinylated ubiquitin and recombinant wild-type H3N-GST (WT) and arginine-substituted H3N-GST mutants as substrates. Proteins were analyzed by Western blotting and silver staining. mock: reaction without substrate. Asterisks indicate ubiquitylated H3N-GST proteins.
Fig.Figure 5(A) Distribution of the chromatin-bound levels of all proteins identified by proteomics in U2OS cells treated with protamine (30 μM) or (PR)20 (20 μM) for 90´. Numbers indicate the Pearson´s correlation coefficient (R) and the P-value, which was obtained by a t-test to evaluate the Pearson correlation between both samples. (B) Venn diagram illustrating the overlap among the proteins that show reduced levels on chromatin upon treatment of U2OS cells with protamine (30 μM) or (PR)20 (20 μM) for 90´. The P-value was obtained by a hypergeometric test. (D) Venn diagram illustrating the overlap among the proteins that show reduced levels on mRNA upon treatment of U2OS cells with protamine (30 μM) or (PR)20 (20 μM) for 90´. The P-value was obtained by a hypergeometric test. (C) Distribution of the levels of all proteins identified after isolation of the RNA-bound proteome in U2OS cells treated with protamine (30 μM) or (PR)20 (20 μM) for 180´. RNA-binding proteins (RBPs) were identified as those significantly enriched after the isolation of mRNA-binding factors. Note that treatment with either peptide leads to a selective reduction in the levels of RBPs bound to mRNA that is not observed for the rest of the factors (Not RBPs) (n=2). P values were calculated with two-sided two sample t-tests assuming equal variances. Center lines indicate median values; box limits indicate the 25th and 75th percentiles; whiskers extend 1.5 times the interquantile range from the 25th and 75th percentiles and outliers are represented by dots.
Figure 1A. Schematic structure of pRb, location of the 14 CDK sites and the four Ser/Thr-to-Ala substitutions (denoted by *) in Rb∆K4 knock-in mice in exon 23. Clustering of phosphorylation sites based on protein interaction profiles of monophosphorylated pRb species. N, N-terminus; A-B, pocket domain; L, spacer/linker; C, C-terminus which includes exon 23. B. Western blot analysis of Rb∆K4 thymocytes with an antibody that recognizes both hyper-phosphorylated (ppRb) and hypo-phosphorylated pRb species. C. Representative flow cytometric profiles of thymocytes from 6-week old Rb∆K4 and control mice for CD3-, CD4- and CD8-positive cell populations. Bottom, average of CD3-, CD4- and CD8 single- and double-positive cells in Rb∆K4 vs. control thymocytes showing no significant (n.s.) difference by student's t-test. Bars represent mean ±SD (standard deviation); n=6 biological replicates). D. Short telomeres in Rb∆K4 mice. Images of splenocyte metaphase spreads hybridized with the telomere repeat probe (pink). Note shorter telomeres in Rb∆K4 splenocytes (arrows). Bottom, quantitative analysis of 10 splenocyte metaphases from one pair of Rb∆K4 and control littermate; (P<0.001 by two-tailed Mann-Whitney-Wilcoxon test).
Figure 2(a) Cell surface VEGFR2 levels detected by cell surface biotinylation, using thiol-cleavable sulfo-NHS-SS-biotin, of HDMEC transfected with PALD1 siRNA (#1 and #2) or non-targeting control ("c") siRNA, followed by VEGF-A stimulation (50 ng/ml) for indicated time periods. Total lysates (input) and streptavidin (SA) pull down, immunoblotted for VEGFR2, Paladin, and actin. 'No biotin ctrl', cells not treated with sulfo-NHS-SS-biotin. (b) Quantification of data in (a). VEGFR2 surface levels (data pooled for the indicated time points) normalized to total VEGFR2 levels and compared between control and siRNA treated HDMEC. n=4 for each time point, biological replicates, Mean±SEM. Data information: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. (c) Internalized pool of VEGFR2 after VEGF-A treatment (50 ng/ml) of non-transfected HDMEC ("NT") or HDMEC transfected with PALD1 siRNA (#1 and #2) or non-targeting control siRNA ("c"). Cell surface biotinylation was performed prior to VEGF-A stimulation and at indicated time points, remaining cell surface biotin was stripped and the internalized pool of VEGFR2 was collected by SA pull down. Immunoblotting of the total lysate (input) and SA pull down fraction for VEGFR2, Paladin and actin. (d) Quantification of data in (c). Data were normalized to total VEGFR2 levels in the lysate after subtraction of signals in biotinylated and stripped samples. Mean±SEM, unpaired t-test for indicated time points, normalized to cntrl siRNA sample. n=3 for each time point, biological replicates. Data information: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. (e) Analysis of EEA1 and VEGFR2 vesicles following PALD1 knockdown. Representative images of VEGFR2 (green)/EEA1 (red) double-positive (yellow) vesicles in negative control siRNA, and PALD1 KD#2 siRNA-silenced HDMEC at 0, 2 and 10 min of VEGF-A stimulation (50 ng/ml). DAPI in blue, scale bar: 10 µm. Inset shows only EEA1 channel, scale bar: 10 µm. (f) Quantification of (e), number of EEA1 positive (top) or VEGFR2-EEA1 double-positive vesicles (bottom) per field of view. Mean±SEM, Two-way ANOVA, n=3 biological replicates. Data information: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. (g) HDMEC stained for PI(4,5) P2 (cyan), VE-cadherin (red) and Paladin (yellow in inset) following treatment using negative control or PALD1 (KD#2) siRNA. VEGF-A stimulation for 0, 2 or 10 minutes (50 ng/ml). DAPI in blue. Scale bar: 10 µm (h) Quantification of (g), Total PI(4,5) P2 signal (top) or intracellular PI(4,5) P2 not overlapping with VE-cadherin (bottom). Mean±SEM, two-way ANOVA, n=3 biological replicates. Data information: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
Figure 1A, Experimental workflow of nonCM isolation from zebrafish hearts and scRNA-seq (10x Genomics). B, scRNA-seq data of adult zebrafish cardiac nonCM visualized on tSNE and colored by cell types. EC, endothelial cells; FB, fibroblasts; MC, macrophages; Mes, resident mesenchymal cells; T/NK/B, T/NK/B cells; Neutro, neutrophils; Eryth, erythrocytes; Throm, thrombocytes. C, Violin plots showing expression of canonical markers for each cell type. GO analysis (DAVID) of upregulated genes in each population was performed and representative GO terms were listed on the right. D-F: NonCMs expressing canonical EC markers in panel b were zoom-in analyzed with LIGER. D, Cells visualized on tSNE and colored by cell types. eEC, endocardial EC; lEC, lymphatic EC; cEC, coronary EC; Mural, mural cells. E Pie chart showing contribution of each cell type. F Expression of newly identified markers of each EC subpopulation shown on tSNE. G, Pie chart showing nonCM composition (erythrocytes and thrombocytes excluded). H, Dotplot showing expression of top 8 positive markers identified for each nonCM population.
Figure 6A wa3 hearts express higher level of Il1 than littermate WT hearts without significant changes in selected structural and regulatory genes as myosin binding protein C (Mybpc3), phospholamban (Pln), ryanodine receptor 2 (Ryr2), and sarcoglycan gamma (Sgcg). Hearts removed from perfused mice and mRNA was subjected for q PCR. Difference between knockdown and control with p values = 0.03 (Student's t test) is indicated with an asterisk. B. A scatter plot showing the deviation from normal expression by highlighting the differentially expressed genes of newborn, 7-week- and 12-week- hearts (p<0.05 threshold, calculated by Deseq algorithm "R"). The expression level of s100a9 was higher in wa3 mice than in WT by 2.8 times. For the full list of genes see Tables S1 and S2, and functional analyses in Figures S3-S6. C. Hierarchical clustering heat map of differentially expressed genes in the hearts of 7- and 12- week old wa3 mice in comparison to WT.