EMBO/sd-character-level-ner huggingface dataset:
Global transcriptome analysis reveals that mitochondria are significant contributors to the development of the senescent phenotype, particularly the SASP.Figure 2A) Column clustered heat-map showing all genes which are differentially expressed between proliferating (Prol), senescent (Sen) and mitochondria-depleted senescent (MDSen) Parkin-expressing MRC5 fibroblasts (FDR≤5%, absolute fold ≥2). Genes are by column and samples by row. The colour intensity represents column Z-Score, where red indicates highly and blue lowly expressed; B) Venn diagram showing the overlap of the differentially expressed genes (FDR≤5%, absolute fold ≥2) between proliferating and senescent cells, and between senescent and MD senescent cells. Direction of change is indicated; C) Table of the 10 most significantly enriched (FDR ≤5%) gene ontologies for the "fully reversed" genes, as defined by the tool DAVID. Ontologies which are amongst the 10 most significantly enriched for genes that are significantly up-regulated between proliferating and senescent cells are indicated in red; D) Column clustered heat-maps of all SASP genes and cell-cycle genes which are differently expressed in senescence; genes are by column and samples by row. The colour intensity represents column Z-Score, where red indicates highly and blue lowly expressed. SASP heatmap: genes that are labelled in green represent the 60% that are fully reversed and genes labelled in pink represent the 5% that became exacerbated upon mitochondria clearance. Cell Cycle heatmap: genes labelled in red are significantly changed upon mitochondria clearance.
Figure 4BF and IFM images of control and Rab22A-depleted melanocytes. The colocalization coefficient (r, in mean±SEM) between the two markers indicated separately. Nuclei are stained with Hoechst33258 ), arrows indicate the melanocyte pigmentation and arrowheads point to the localization of TYRP1/GFP-Rab22A. Scale bars: 10 μm Graph represents the quantified melanin content in control sh, Rab22A sh and BLOC-1- melanocytes. n=3. The fold-change in melanin content (mean±SEM) indicated separately Immunoblotting analysis of proteins in Control sh and Rab22A sh melanocytes ), protein band intensities were quantified and indicated on the gels EM of control and Rab22A-knockdown MNT-1 melanocytes. Arrowheads indicate PMEL fibrils. Scale bars: 1 μm. I, II, III, IV: stages of melanosomes. M: mitochondria. Magnified view of insets are shown separately and they emphasize the stage I melanosomes in both conditions. Note: the presence of hybrid stage I/II melanosomes in Rab22A-knockdown cells. Insets (b)-(d) and (g)-(h) are from different cells of respective condition. Scale bars: 200 nm. BF and IFM images of GFP-Rab22A transfected BLOC-1-(Mu) melanocytes ), arrows indicate the melanocyte pigmentation and arrowheads point to the localization of TYRP1/GFP-Rab22A. Scale bars: 10 μm Immunoblotting analysis of proteins in BLOC-1- and BLOC-1R melanocytes protein band intensities were quantified and indicated on the gels
Figure 2(A) The snf1Δ and snf4Δ mutant cells are hypersensitive to HU. Five-fold serial dilutions of the overnight cultures were assayed on normal growth media (YPD) with or without 200 mM HU. A plate in which 2% glucose was replaced by 2% non-fermentable carbon (galactose) was applied as a control. All plates were cultured at 30°C for 48 h before photography. (B) Rad53 hyperphosphorylation of WT and snf1Δ in response to HU. Cells were synchronized in G1 and released into the fresh medium containing 200 mM HU for the indicated time. Cell extracts were prepared for immunoblotting against anti-Rad53 antibodies. Tubulin was probed as a loading control. (C) Rad53 phosphorylation in various snf1 alleles in response to HU. The plasmids expressing the indicated snf1 alleles were transformed into snf1Δ. Rad53 phosphorylation was detected as above. (D) The snf1 kinase-defective mutants are sensitive to HU. Yeast spot assays were performed as in Fig 2A. The phospho-mimetic snf1T210D (a constitutive active mutant) was applied as a control for nonphosphrylable snf1T210A. (E) H2A S129 phosphorylation (namely γ-H2A) of WT and snf1Δ in response to HU. γ-H2A was detected by immunoblotting. (F, G) HU induces a rapid Snf1 T210 phosphorylation. Exponential cells were treated by 200 mM HU for the indicated time. Protein extracts were prepared as described in the Methods and Materials. Three independent experiments were done. Quantitation of Snf1 T210 and Snf1 signals was performed by Image J. The relative ratio of Snf1 T210/Snf1 was shown in (H). Error bars represent standard deviations (SD) from three biological repeats.
a) Confocal analysis showing the effects of PLD inhibition (FIPI, 750 nM) on the levels of endogenous LC3.f6(a) Confocal analysis showing the effects of PLD inhibition (FIPI, 750 nM) on the levels of endogenous LC3. CHO cells were treated with PLD inhibitor FIPI or DMSO for 3 h in the presence of serum (normal medium, Nm) or in the absence of nutrients (starvation, St). Insets denote high magnification of the selected areas. Scale bar, 10 μm. (b) Quantification of the average number, size and surface area of LC3-positive compartments per cell after FIPI treatment. Black bars, DMSO (vehicle); grey bars, FIPI treatment. Values denote means±s.e.m. (n=180-285 cells for Nm; n=449-519 cells for St). **P0.01. (c) Western blot analysis showing levels of LC3-I and LC3-II in control- and FIPI-treated CHO cells grown in normal medium (Nm), after nutrient starvation (St) or after nutrient starvation in the presence of 50 nM bafilomycin (St+B). Treatments were for 3 h. Right panel: quantification of protein signal intensities showing LC3-II protein levels in extracts from nutrient-starved and bafilomycin-treated CHO cells following FIPI treatment. Black bars, DMSO (vehicle); grey bars, FIPI treatment. Values denote means±s.e.m. (n=4). (d) Western blot analysis of the effects of PLD inhibition on the levels of total Tau, phosphorylated Tau (p-Tau) and detergent-insoluble Tau. Organotypic brain slices from hTau mice were treated with either DMSO (D) or 750nM FIPI (F) and harvested after 5 days of treatment. Left panel: western blot analysis showing immunoreactivities for total human Tau (CP27), hyperphosphorylated tau (p-Tau, ser202/ser205, CP13), p62 and tubulin from the supernatant fraction and aggregated tau (CP27) and p62 from the sarkosyl-insoluble fraction. Two representative pairs are displayed. Right panel, quantification of total proteins (Tau, p-Tau, p62 and tubulin) and sarkosyl-insoluble aggregates of tau (insoluble Tau) and p62 (insoluble p62). Black bars, DMSO (vehicle); grey bars, FIPI treatment. Values denote means±s.e.m. (n=7). **P0.01; *, P0.05. For the statistical analysis of FIPI-treated samples, tau and p62 protein levels were compared with tubulin.
Figure 3(A) Representative image of 5-day-old cest-2.2p::GFP (left panel) and an age-matched GFP-negative control (right panel). Scale bar= 100 μm. (B) Schematic representation of CEST-2.2 participation in very long chain fatty acid (VLCFA) metabolism and ascaroside biosynthesis. Metabolites from peroxisomal β-oxidation may contribute to the biosynthesis of medium-chain fatty acid (1) as well as intracellular signaling (2). Ascarosides may take part in cell-autonomous and/or -nonautonomous signaling. (C) Confocal images of mitochondrial morphology in the intestines of gas-1(fc21) animals exposed to either 1.7% ethanol (control), 1 μM ascr#8 or 1 μM ascr#81 (scale bar= 5 μm). (D) Representative lifespan analysis of gas-1(fc21) animals exposed to ethanol (control) and 1 μM ascr#8 from hatching. Underneath is the average median lifespan ± SEM across replicates. Data information: the median lifespan ± SEM is reported underneath the graphs and additional information (e.g., n numbers) (E) qRT-PCR of cest-2.2 expression in the intestines of 5-day-old wt, gas-1(fc21), gas-1(fc21); cest-2.2 O/E and nuo-6(qm200) animals (one-way ANOVA with Tukey's multiple comparison test, n=3 biological replicates). Data information: points represent mean± SEM. Across experiments, p value summary is ns= not significant, *p<0.05, **p < 0.01; ***p < 0.001, ****p<0.0001. (F) Volcano plot of transcriptomes of intestines from gas-1(fc21) vs. wt (left panel) and gas-1(fc21); cest-2.2 O/E vs. gas-1(fc21) mutants (right panel). In red: significantly dysregulated genes (5% FDR). (G) Pie charts of differentially regulated genes in gas-1(fc21) vs. wt (top panel) and gas-1(fc21); cest-2.2 O/E vs. gas-1(fc21) mutants (bottom panel), showing proportion of genes of unknown function or involved in metabolism. Genes involved in lipid metabolism specifically represented in a small box within the metabolism slice. (H) Heatmap of genes involved in lipid metabolism in gas-1(fc21) vs wt and gas-1(fc21); cest-2.2 O/E vs gas-1(fc21). (I) Ingenuity Pathway Analysis (IPA) of transcriptome in gas-1(fc21) vs. wt (top panel) and gas-1(fc21); cest-2.2 O/E vs. gas-1(fc21) (bottom panel) with their predicted consequences on lipid metabolism. In red/green: results from the transcriptome. In blue/orange: IPA-generated predicted outcome of this gene dysregulation. Shades of red and green are proportional to log2(fold change), while shades of blue and orange are proportional to the statistical strength of the prediction (z-score), as calculated by IPA.
Figure 2Dose-dependent viability assays of BRCA1-proficient (+BRCA1) or -deficient (-BRCA1) human RPE1-hTERT cells treated with drugs at the indicated concentrations for six days. Dose-dependent viability assays of BRCA1-proficient (+BRCA1) or -deficient (-BRCA1) TP53-deleted cells treated with drugs at the indicated concentrations for six days. Dose-dependent viability assays of Brca1+/+ and Brca1-/- mouse mammary tumour-derived cell lines treated with drugs at the indicated concentrations for six days.
scRNA-seq reveals differences in expression characteristics of TATA and paused genes.Figure 3(A) The differences in cell-to-cell gene expression variability for the TATA and the paused effector gene groups were analyzed using the scRNA-seq data. The coefficient of variation (standard deviation/mean) of gene expression was calculated for all genes in the tissue with the highest expression for each gene. The median coefficient of variation was consistently lower for the paused genes compared to the TATA genes. (B) The frequency of cells with any detectable expression (> one read) was calculated in tissues with the highest expression for each gene (expressing tissue) and in five other tissues with the least expression for each gene (other tissues). The frequency of cells with detectable expression in the expressing tissues, a measure of expression robustness, is lower for the TATA genes compared to the highly paused genes (left) (Wilcoxon two-sided test, *P <10-15). The frequency of cells with detectable expression in the other tissues, a measure of background expression, is also lower for the TATA genes compared to the paused genes (right) (Wilcoxon two-sided test, *P <10-15). (C,D) Normalized gene expression levels (read count normalized for each cell, log2) in different tissues, from the scRNA-seq experiment, for a TATA group gene, and a highly paused group gene, are shown. (C) The TATA gene, Ccp84Aa, shows noisy expression in the epidermis, without detectable background expression in non-expressing tissues. (D) The highly paused gene, Gip, shows very robust expression in crystal cells, but has high background expression in the non-expressing tissues. (E) The number of annotation terms associated with each gene in the BDGP in situ database. This is a measure of whether the expression of a gene is restricted to specific subsets of tissue (Wilcoxon two-sided test, *P <10-15). (F) The coefficient of variation across different isogenic lines of Drosophila from DGRP, after being corrected for dependence on expression (loess regression) is plotted for different effector gene groups (Sigalova et al. 2020). Genes from the TATA enriched group show high variability compared to the paused genes (Wilcoxon two-sided test, *P <10-15). Boxplots in all panels show the median as the central line, the first and the third quartiles as the box, and the upper and lower whiskers extend from the quartile box to the largest/smallest value within 1.5 times of the interquartile ran
Netrin-3 is a target gene of NeuroD1 and ASCL1 in SCLC.Figure 5A. ChIP-seq analysis of NeuroD1 and ASCL-1 binding on the netrin-3 gene promoter. A major binding site was detected (red and blue peaks for NeuroD1 and ASCL1 respectively). Input controls are shown below (black). B. ChIP-seq analysis of NeuroD1 and ASCL-1 chromatin enrichment, which was associated with broad H3K27ac-enriched regions at the netrin-3 locus (red line), indicating a transcriptionally active region in SCLC cell lines. (rep= experimental replicates). C. Q-RT-PCR analysis of netrin-3 gene expression in NCI-H82 (n=6) and NCI-H69 (n=4) SCLC cell lines after Neurod-1 and ASCL-1 silencing by siRNAs (U-test). Error bars indicate s.e.m.
Glomerular PAI-1 expression correlates with glomerular senescence and predicts outcomes of kidney transplant from elderly donors.Figure 7A: PAI-1 staining (left panel) and quantification (right panel) in glomeruli of kidney biopsies from young (< 40 years of age, n = 8) or old (> 80 years of age, n = 10) kidney donors at time of transplantation (M0). Original magnification X400. Scale bar = 20 μm. B: p16 staining (left panel) and quantification (right panel) in glomeruli of kidney biopsies from young (< 40 years of age, n = 8) or old (> 80 years of age, n = 10) kidney donors at time of transplantation (M0). The arrows point to the staining. Original magnification X400. Scale bar = 20 μm. Data information: Data are means ± SEM. Statistical analysis: t-student test C: PAI-1/CD34 coimmunostaining in old- positive or negative glomerular PAI-1 donors. Original magnification X630. Scale bar = 10 μm. D: p16/CD34 coimmunostaining in old-positive glomerular PAI-1 donors. Original magnification X630. Scale bar = 10 μm. E: Representation of PAI-1 and p16 staining results at time of transplantation in young (n = 8) or old donors (n = 10). Each line represents one patient. Green color is used for negative staining, red color for positive staining. F: Quantification of the glomerulosclerosis score at time of (M0), or 12 months after (M12) kidney transplantation from elderly donors in patients displaying a negative (PAI-1-, n = 17) or positive (PAI-1+, n = 13) glomerular PAI-1 staining at time of transplantation (M0). Data information: Data are means ± SEM. Statistical analysis: ANOVA followed by the Tukey-Kramer test G: Estimated glomerular filtration rate (eGFR) at time of (M0), or 12 months after (M12) kidney transplantation in patients displaying a negative (PAI-1-, n = 17) or positive (PAI-1+, n = 18) glomerular PAI-1 staining at time of transplantation (M0). Data information: Data are means ± SEM. Statistical analysis: ANOVA followed by the Tukey-Kramer test H: Urinary PAI-1 measured by ELISA in a cohort of elderly patients with (n = 48) or without (n = 48) chronic kidney disease. Data information: Data are means ± SEM. Statistical analysis: t-student test
The interaction between FYCO1 and LC3B is mediated by a short conserved acidic motif C-terminally from the FYVE domain in FYCO1 and both N- and C-terminal domains of LC3B.Figure 2.(A) Schematic structure of FYCO1 and its homologous proteins RUFY1b and EEA1. In the multialignment sequence, identity is indicated by black highlighting, and residues highlighted in gray indicate substitutions to chemically similar amino acids in more than 50% of the compared sequences. (B) The region of FYCO1 between aa 1,276 and 1,294 is essential and sufficient for the interaction with LC3B. GST or GST-LC3B were incubated with [S35]methionine-labeled deletion mutants of FYCO1 and processed as in Fig. 1 C. (C) LIR is essential for colocalization of FYCO1 with LC3B in HeLa cells. HeLa cells expressing the indicated constructs were imaged by confocal microscopy. Insets show an enlarged field of interest. (D) Both N- and C-terminal domains of LC3B are needed for efficient interaction with FYCO1. GST, GST-LC3B, or its deletion mutants were incubated with [S35]methionine-labeled myc-FYCO1 and processed as in Fig. 1 C. ARG, autoradiography; CB, Coomassie blue. (E) The interaction between FYCO1 and LC3B is direct. GST or GST-LC3B was incubated with recombinant MBP-FYCO1863-1,478. Protein complexes were isolated and visualized by immunoblotting with anti-GST or anti-MBP antibodies. WB, Western blot. Bars: (C) 10 µm; (C, insets) 1 µm.
Figure 5A, 4T1 cells were pretreated with AZD6738 at indicated concentrations for 2 hours, and then irradiated with 10 Gy. 4 days later, Ifnb1 mRNA level was measured by real-time quantitative PCR. Data are presented as mean + standard error of the mean of three biological replicates. B, Ifnb1 mRNA levels were measured by real-time quantitative PCR in WT, Sting KO, Mavs KO, and Irf3 KO 4T1 cells. Data are presented as mean + standard error of the mean of three biological replicates. C, MC38 cells were pretreated with AZD6738 at indicated concentrations for 2 hours, and then irradiated with 20 Gy. 3 days later, Ifnb1 mRNA level was measured by real-time quantitative PCR. Data are presented as mean + standard error of the mean of three biological replicates. D, Ifnb1 mRNA levels were measured by real-time quantitative PCR in WT, Sting KO, Mavs KO, and Irf3 KO MC38 cells. Data are presented as mean + standard error of the mean of three biological replicates.
Figure 1AGS cells were infected with H. pylori or treated with 20 ng/ml LTα1β2 for the indicated times. Total cell lysates were analyzed by immunoblotting using the indicated antibodies. AGS cells were infected with H. pylori P1 wild‑type or isogenic ∆HP0857 (mutated in the gmhA gene) strains. Total cell lysates were analyzed by immunoblotting using the indicated antibodies. AGS cells were treated with 200 nM synthetic ADP‑heptose for the indicated times. Total cell lysates were analyzed by immunoblotting using the indicated antibodies. AGS cells were pre‑incubated with 100 μM dynamin GTPase inhibitor Dynasore for 60 min followed by H. pylori infection or addition of ADP-heptose (200 nM). Total cell lysates were analyzed by immunoblotting using the indicated antibodies. CagA demonstrated a similar infection rate.
Figure 5BiFC analyses of interactions between VISP1 and SGS3 or RDR6 in N. benthamiana leaves at 60 hpi. RbcL served as a negative control. Scale bar = 20 μm. Schematic representation of the Arabidopsis SGS3 protein and deletion mutants. Deleted regions are indicated by dashed lines. N: N terminus including Zinc finger (ZF), XS: Rice protein X and SGS3, CC: Coiled-coil domain. Schematic representation of VISP1 substitution mutants in the UIM and ARM regions. UIM: ubiquitin-interacting motif, ARM: Arginine/lysine-rich motif. BiFC assays analyzing interactions between VISP1 and SGS3 or its deletion mutants in N. benthamiana leaves. Scale bar = 20 μm. BiFC assays analyzing interactions between SGS3 and VISP1, VISP1mUIM or VISP1mARM in N. benthamiana leaves. Scale bar = 20 μm. Co-immunoprecipitation analyses of the interactions between SGS3, SGS3ΔCC, or SGS3CC and VISP1 or VISP1mARM. N. benthamiana leaves were agroinfiltrated with constructs as indicated and treated with 2 mM 3-MA at 48 hpi, and collected for IP with anti-Flag beads 12 h later. GST pull-down analysis of the in vitro interactions of SGS3CC with VISP1, VISP1mARM or VISP1mUIM. GST- VISP1, GST- VISP1mARM, GST- VISP1mUIM or GST were incubated with SGS3CC-His for IP with anti-GST beads, and detected by Western blotting with GST or His antibodies.
Fig.Figure 6Fig. 6 CryoEM structure of fatty acid synthase resolved to 4.7 Å as obtained from cryo electron micrographs of fraction numbers 7-9. (A) The cryoEM of C. thermophilum fatty acid synthase (ctFAS) map is shown isosurface rendered and superimposed with the fitted X-ray structure of yeast FAS (Jenni et al, 2007). The domes and the cap show the unambiguous fit of α-helices and β-strands. (B) A slice through the central wheel of fungal FAS. The pitch of α-helices is resolved. (C) As for (B) but sliced through the dome structure. (D) Location of acyl carrier protein (ACP) within the cryoEM map of ctFAS and the position of additional density outside the dome. (E) Fit of ACP in the cryoEM map of ctFAS and comparison with the crystallographically determined location in yeast FAS; additional density within the active site, possibly resembling the acyl chain bound on the ACP is observed. (F) Molecular model of the interaction between the ACP and the ER domains of ctFAS in cartoon representation. The model was derived from a rigid fit from (B) and subsequently flexibly refined for clash removal and interface energetics optimization.
Fig.Figure 6A: Cal27 and JHU029 cells were transfected with control or ELDR siRNA and relative expression of miR-7 was analyzed by qRT-PCR. U47 was used as an internal control. n = 3 biological replicates. Data are represented as the mean ± SD, B: Cal27 and JHU029 cells were transfected with control or ELDR plasmid and relative expression of miR-7 was analyzed by qRT-PCR. U47 was used as an internal control. n = 3 biological replicates. Data are represented as the mean ± SD, C: Cal27 and JHU029 cells were transfected with control or mimic miR-7 and relative expression of ELDR was analyzed by qRT-PCR. 18S rRNA was used as an internal control. n = 2 biological replicates; n= 3 technical replicates. Data are represented as the mean ± SD, D: Cal27 and JHU029 cells were transfected with miR-7 mimic, ELDR plasmid or both miR-7 and ELDR plasmid. Cell lysates were analyzed by Western blot analysis for EGFR expression using specific antibody. The membrane was reprobed with an antibody against Actin as an internal control. Representative bands were cropped from same gel. Right panels show quantitative representation of Western blot band intensities. n = 3 biological replicates. Data are represented as the mean ± SD, E: Relative expression of miR-7 in Cal27 and JHU029 cells compared to NOK cell line analyzed by qRT-PCR. U47 was used as an internal control. n = 3 biological replicates and 3 technical replicates. Data are represented as the mean ± SD, F: Schematic representation of ELDR and miR-7 mediated regulation of EGFR signaling. Sharp arrow indicates activation and blunt arrow indicates inhibition.
Figure 1(A) Domain diagram of CK1δ orthologs from S. cerevisiae, S. pombe, and M. musculus, with percent sequence identity (% ID) noted, and of S. cerevisiae Mam1. Hrr25 orthologs from S. cerevisiae and its close relatives possess a 'central domain' (green) and a C-terminal proline/glutamine-rich region predicted to be disordered in solution (magenta). (B) Phylogenetic tree constructed from an alignment of 93 fungal Hrr25 orthologs. Branches in green represent Hrr25proteins from Saccharomycotina yeast with the conserved central domain (see Figure EV1A for sequence alignment). Branches in blue represent Saccharomycotina Hrr25 proteins lacking this domain. Orange stars indicate the presence of a Mam1 ortholog in each species' genome. E. cymbalariae (white star) possesses an un-annotated Mam1 ortholog on chromosome VII (region ~313000-315000, 32% identity to S. cerevisiae Mam1 residues 83-242, identified by TBLASTN (NCBI)). Purple diamonds denote organisms in which point-centromere sequences have been identified (Meraldi et al, 2006; Gordon et al, 2011). Point-centromeres and Mam1 orthologs are both exclusive to the group of yeast whose Hrr25 orthologs possess the central domain.