EMBO/sd-character-level-ner huggingface dataset:
Figure 8(A) A549 cells of the indicated genotypes were treated for 24h as indicated (CPT = camptothecin, ZM = ZM447439). Samples were subjected to immunoblotting; n = 3 independent experiments. (B) A549 cells treated as in (A) were subjected to fluorescence microscopy and centrosome abundance was assessed by visually scoring γ-tubulin-positive centrioles per cell. N = 3, ≥ 50 cells from each independent experiment. (C) RT-qPCR analysis of PIDD1 mRNA expression in A549 cells upon treatment with increasing doses of Nutlin 3a (i.e. 3.3 μM or 10 μM) using two independent probes. The average fold of induction ± standard deviation is shown. N = 3 biological replicates with two technical replicates each. Comparisons were performed between treatments of every genotype and the corresponding treatment of wild type (WT) cells with increasing doses of Nutlin 3a (i.e. 3.3 μM or 10 μM) using two independent probes. (D) Immunoblot analysis of samples treated N = 3 independent experiments. (E) Proposed model for the centrosome-dependent PIDDosome activation upon different stimuli. The centrosome constitutively acts as PIDD1 centralizer. A local increase in PIDD1 concentration (achieved either by centrosome clustering or upon p53 activation) triggers PIDDosome activation. In ANKRD26-deficient cells, the inability of the centrosome to generate a local increase in PIDD1 concentration hinders the activation of the complex in response to both stimuli.
Figure 3a, Polarised gene regulatory states underlying stromal subclasses. Heatmap shows the averaged regulon activity (area under the curve; AUC) for the top 50 highest TFs regulons as estimated using SCENIC. All regulons are statistically enriched across the four subsets (p < 1x10-5 One-way ANOVA). Heatmap is clustered using Euclidean distance and complete linkage. b, Candidate transcriptional drivers of each CAF and PVL subset. Violin plots showing the log normalised gene expression (left) of the TF and its respective AUC regulon activity (right). TFs ZEB1 and FOXP1 enriched in myofibroblast-like CAFs, EGR2 and TCF7L2 enriched in inflammatory-like CAFs, MEF2C enriched in PVL cells and NR2F2 enriched in immature-PVL cells.
Figure 8A) Growth sensitivity to MMS and phleomycin of a Cdk phospho-deficient mutant of Spc110. Ten-fold serial dilutions from overnight cultures of wild-type, spc110S36-91A and a cdc14-1 cells grown on solid rich media containing mock DMSO, MMS or Phleomycin at 30ºC. B) Diagram showing the genomic background and the approximation used to measure DSB-SPB interaction. Spc110 and spc110S36-91A were labelled with the RFP in a background containing Ddc2-GFP as DSB reporter. D1: inter-SPB distance; D2: DSB distance to proximal SPB; D3: DSB distance to distal SPB. At least 100 cells from three independent experiments were scored using the maximum projection of nine z-planes. Graph shows the mean ± S.D. P values were calculated using a 2-tailed unpaired Student t test. C) Time-lapse experiments of living cells expressing the HO endonuclease in wild-type and spc110S36-91A mutants cells. Five z-planes every 5-second intervals over a period of 5 minutes were captured. Quantifications were made using the average SPB track length and velocity of at least 100 cells. Spc110 and spc110S36-91A were labelled with the RFP and used as SPB reporter. D) Southern blot of wild-type and spc110S36-91A mutant harbouring the inter-chromosomal gene conversion assay portrayed. An overnight culture was induced by adding galactose at 32ºC to express the HO endonuclease. Samples were collected at different time-points, genomic DNA was extracted, digested with EcoRI and analysed by Southern blot. Blots were probed with a MATa-only and actin (for loading control) DNA sequences. Graphs show quantification of gene conversion, DSB induction and crossover vs non-crossover ratio. All data were normalized using the actin signal. Graphs represent the mean ± S.D. from three independent experiments. P values were calculated using a 2-tailed unpaired Student t test. Asterisk denotes an overexposed film to visualize the formation of crossover products. D) Southern blot of wild-type and spc110S36-91A mutant harbouring the inter-chromosomal gene conversion assay portrayed. An overnight culture was induced by adding galactose at 32ºC to express the HO endonuclease. Samples were collected at different time-points, genomic DNA was extracted, digested with EcoRI and analysed by Southern blot. Blots were probed with a MATa-only and actin (for loading control) DNA sequences. FACS profiles for DNA content are included. Graphs show quantification of gene conversion, DSB induction and crossover vs non-crossover ratio. All data were normalized using the actin signal. Graphs represent the mean ± S.D. from three independent experiments. P values were calculated using a 2-tailed unpaired Student t test. Asterisk denotes an overexposed film to visualize the formation of crossover products.
Figure 6(A) analysis using transcriptomic data from the liver of sepsisBIM mice (16h after intraperitoneal injection of live E. coli). (B-C) Enrichment plots from GSEA performed using LIVER-ID genes (B) or the response to ERS gene set (GO:0034976) (C) as the gene set and transcriptomic differences in sepsisBIM vs Control mouse liver as the ranked gene list. (D) Comparison of the transcriptome of the indicated liver injury models with that of the developing mouse liver PC1 is the first principal component which represents 63.55% of the variability within the mouse liver differentiation study. PC1 was used to project the liver injury studies. APAP, Acetaminophen overdose - Model of drug-induced acute liver injury. CCL4, Carbon tetrachloride hepatotoxicity - Model of drug-induced chronic liver injury. MCD-HF, Methionine-choline deficient diet with high fat - Model of NASH/fibrosis. (E) Box plots showing Log2 FC for ERS UP, DOWN or unchanged genes in sepsisBIM vs Control mouse liver (6 mice per group). Box plots are composed of a box from the 25th to the 75th percentile with the median as a line and min to max as whiskers. One-way ANOVA with Welch's correction and Dunnett's Modified Tukey-Kramer pairwise multiple comparison test was used to assess statistical significance, *P < 0.05. (F) RT-qPCR analyses of selected ERS UP genes and LIVER-ID TFs monitoring expression changes in the liver of sepsisBIM vs Control mice (6 mice per group). The bar graph shows means ± SD (standard deviations). One-sample t-test with BH correction for multiple testing was used to determine if the mean Log2 FC sepsisBIM/Control is statistically different from 0, *P < 0.05. (G) Nuclear extracts from livers of control or sepsisBIM mice were subjected to Western blot or Simple Western immunoassay with antibodies against HNF4A, NR1H4 or FOXA2. LMNA was used as loading control. (H) Nuclear extracts from livers of sepsisBIM mice pre-treated for 4 consecutive days with vehicle or 500 mpk TUDCA were subjected to Western blot with antibodies against HNF4A, NR1H4, FOXA2 or XBP1S. TFIIB was used as loading control. (I) Densitometric quantification of the protein expression data from 6 mice precondition shown in panel (H) (3 mice per condition) The bar graph shows means ± SD (standard deviations). One-sample t-test with BH correction for multiple testing was used to determine if the mean Log2 FC TUDCA/Vehicle is statistically different from 0, *P < 0.05. (J) Serum alanine aminotransferase (ALT) (left) and aspartate aminotransferase (AST) activities (right) from sepsisBIM mice pre-treated for 4 consecutive days with vehicle or 500 mpk TUDCA (10 mice per group). The bar graphs show means ± SD (standard deviations). Student's t-test was used to assess statistical significance, *P < 0.05.
Description of the phosphoproteomic data set and its generation.Figure 1a Experimental workflow to quantify phosphorylations occurring at the early time points after TCR stimulation. Digested peptides were analyzed by MS before further enrichment to control the impact of TCR stimulation on protein abundances (proteome). Further steps of TiO2 enrichment and phospho-tyrosine immunoprecipitation (pY-IP) were conducted to quantify changes in the phosphorylated serines and threonines (pST phosphoproteome), as well as phosphorylated tyrosines (pY phosphoproteome), respectively (see the Materials and Methods section for more details). Schematic representations of the measured phospho-kinetics are presented on the right. b, c Phospho-sites identified after TiO2 enrichment b and phospho-tyrosine immunoprecipitation (pY-IP) c. Left panel: pie chart presenting the proportion of identified phosphorylated serines (pS), threonines (pT) and tyrosines (pY) in the two data sets. Right panel: cumulative number of unique identified phospho-sites across the biological replicates (3 and 4 independent experiments for the TiO2 and pY-IP, respectively). Phosphorylated serines, threonines and tyrosines are presented individually next to the total number of identified sites (pSTY). The phospho-sites that were not previously reported in any species in the PhosphoSitePlus database (www.phosphosite.org) are reported in isolation in the graph on the right.
ZNRF3 prevents the secondary axis-inducing activity of Hwa A Morphology of classified embryos at tailbud stage.Figure 6A Morphology of classified embryos at tailbud stage. Scale bars: 1mm. B Ratios of embryos in different classes. Oocytes were injected with 5 pg hwa mRNA or hwa10KR mRNA and 100 pg znrf3 mRNA as indicated. Embryos were obtained through Host transfer technology. C Whole mount in situ hybridization of Spemann organizer markers chrd and gsc in gastrula embryos. Embryos were obtained as descripted in (B). Scale bars: 1mm. D Induction of the secondary (2nd) axis by hwa or znrf3 as indicated in Xenopus embryos. One ventral blastomere of 4-cell stage embryos was injected with hwa mRNA (5 pg) or hwa10KR mRNA (5 pg) and znrf3 mRNA (100 pg). Up: Classes of embryos. Down: Ratio of classified embryos. The 2nd axis (red arrowhead). Scale bars: 1mm. E Western blot analysis of Hwa or Hwa10KR in embryos at blastula stage. 1-cell stage embryos were co-injected with hwa (100 pg) or hwa10KR (100 pg) and znrf3 (1 ng) or znrf3ΔRING (100 pg) as indicated. Lysates were immunoblotted with HA, Flag, and α-tub (α-tubulin).
Figure 1A Sequence alignment of GATA and E-box motifs in Alas2 intron 1 or 8 demonstrating conservation among mammals. B DNase hypersensitivity and ChIP-seq profiles for GATA-1 or Pol II occupancy and histone modifications at Alas2 and ALAS2 (accession numbers; GSM912907, GSM912895, GSM1003744, GSM1003753, GSM1014191, GSM923572, GSM946526, GSM935465, and GSM935462). C CRISPR/Cas9 strategy to delete GATA motifs in intron 1 or 8 of Alas2 gene in G1E-ER-GATA-1 cells. PAM: Protospacer adjacent motif. D DNA sequences at Alas2 introns of wild-type (WT) and mutant clones.
CARS is critical for AMPK activation under cystine deprivation conditions (A) Venn diagram showing the overlap of CaMKK2-binding proteins during cystine deprivation and cyst(e)ine metabolism-related proteins.Figure 2(A) Venn diagram showing the overlap of CaMKK2-binding proteins during cystine deprivation and cyst(e) ine metabolism-related proteins. The schematic diagram shows the peptides detected in the results of IP-MS assays of the AHCY and CARS proteins. The CaMKK2-binding proteins are listed in the Table EV3 and the cyst(e) ine metabolism-related proteins are listed in the Table EV4. (B) 293T cells were transfected with HA-CaMKK2 alone or with Flag-CARS for 48 h and then cultured with cystine-deficient medium or complete medium for 8 h. Cell lysates were immunoprecipitated with anti-Flag, and a WB analysis was performed. (C) 293T cells cultured with cystine-deficient medium or complete medium for 8 h were harvested and subjected to immunoprecipitation with anti-CaMKK2, followed by WB analysis with anti-CaMKK2 and anti-CARS. (D) Flag pull-down assays were performed with Flag-EV or Flag-fused CaMKK2 protein and His-CARS purified from E. coli. (E) WB analysis of p-AMPK, p-ACC, p-Raptor and total AMPK ACC, Raptor protein expression in 293T cells transfected with shRNAs targeting CARS that were further treated with cystine-deficient medium for 8 h. Actin served as the loading control. (F) 293T cells were first transfected with shRNAs targeting CARS or NTC, then transfected with HA-AMPKα1 or HA-AMPKα1 and Flag-CaMKK2 for 48 h, and then, they were cultured with cystine-deficient medium for 8 h. Cell lysates were immunoprecipitated with anti-Flag and subjected to Western blot analysis. (G) 293T cells were transfected with shRNAs targeting CARS or NTC, then they were cultured with cystine-deficient medium for 8 h. Cell lysates were immunoprecipitated with anti-AMPKα and subjected to WB analysis with anti-AMPKα and anti-CaMKK2. (H) WB analysis of p-AMPK, p-ACC, p-Raptor and total AMPK, ACC, Raptor, CARS protein expression in 293T cells and RCC4 cells overexpressing Flag-CARS. Actin served as the loading control. (I) WB analysis of p-AMPK and total AMPK protein expression in CARS-overexpressing 293T cells were further transfected with shRNAs targeting LKB1, CaMKK2, or TAK1. Actin served as the loading control. (J) WB analysis of p-AMPK and total AMPK protein expression in CARS-overexpressing 293T cells and RCC4 cells that were treated with 1 µg/ml STO-609 or DMSO for 8 h. Actin served as the loading control.
Central MTOCs originate from spindle poles(A) Live imaging of MTOC and kinetochore dynamics.Figure 2(A) Live imaging of MTOC and kinetochore dynamics. MTOCs (mNG-Cep192, green) and kinetochores (tdTomato-CENP-C, magenta) in B6D2F1 oocytes are shown. Time in h:mm after NEBD. Circles indicate central MTOCs, which are positioned in the middle region of the spindle later than 4 hours after NEBD. Arrows indicate displacement of the MTOCs. Scale bar, 10 μm. (B) Tracking analysis of central MTOCs. In the left graph, the positions of central MTOCs along the spindle axis are shown over time. MTOC positions were used to calculate distance to the spindle equator. In the right graph, lines showing temporal changes in MTOC-equator distance are aligned based on the time when the MTOC reached a position closest to the spindle equator. Horizontal lines at 7 and at −7 μm denote the thresholds used for the definition of the middle and polar regions of the spindle. (C) Central MTOCs originate from spindle poles and transiently stay in the middle region of the spindle. trajectories of central MTOCs (n=25 MTOCs of 8 oocytes from 3 independent experiments) were analyzed and their temporal changes in MTOC-equator distance are shown (Left graph). Central MTOCs were categorized into two groups: (1) ones that came from polar regions of the spindle (n=21, middle graph) and (2) others that stayed in the middle region throughout the period before reaching a position closest to the equator (n=4, right graph). (D) Origin of central MTOCs. (E) Destination of central MTOCs. The tracks of central MTOCs that originated from polar regions were used (n=21 MTOCs). The pole of origin was defined as Pole 1, while the other pole was defined as Pole 2. MTOC positions along the spindle axis (positive values for those closer to Pole 1) over time are shown. Horizontal lines at 7 and at −7 μm denote the thresholds used for the definition of the middle and polar regions of the spindle. Note that central MTOCs moved back to the original pole (Pole 1) or switched their positions to the opposite pole (Pole 2). (F) Destination of central MTOCs that originated from a polar region was categorized as in (E).
Neurotransmitters differentially regulate cortical interneuron migration A.Figure 5A. Various neurotransmitter genes as identified in 3E. Genes are marked (color-code) to represent the neurotransmitter receptor they encode for: Glycine (Red), Glutamate (Purple), GABA (Yellow) and Serotonin (Blue). B. Simplified schematic representation of the modulation of neurotransmitters in migrating cortical interneurons. GABA, glutamate, glycine and serotonin transmission was modulated. Receptors and their corresponding inhibitors (red arrows) or stimulators (green arrows) are listed for each neurotransmitter system. C. Heatmap depicting the control-normalized values (log2-scaled) for selected parameters (from Figure S9A) for the 8 receptors analyzed in 5B. Grey values indicated non-significant values. In total 3979 moving cell tracks with the following number of tracks per group: Control - 696, Bicuculline-Methiodide - 1189, Saclofen - 527, TPMPA - 138, AP-5 - 429, CNQX - 500, Strychnine - 98, Serotonin - 180, and SR57227 - 222, were analyzed. D. Simplified schematic representation of the effect of the analyzed neurotransmitters (Figure 5B) on migrating cortical interneurons. The effect of each receptor modulation is listed for each neurotransmitter system. The green arrows indicate a stimulatory while red arrows indicate inhibitory effects.
Impaired Ca2+-regulated exocytosis in Trpml1-/- pancreatic acini.Figure 1(a, b) Isolated acini from wild-type (WT) and Trpml1-/- mice (ML1-/-) were used to determine the frequency of Ca2+ oscillations in response to stimulation with 3 and 10 pM CCK (a) and the response to high concentrations of CCK (b). The average frequency from multiple experiments is given in the columns as mean±s.e.m in (a). The traces and columns in (b) show the mean±s.e.m of 3 experiments each includes 4-6 acini composed of 6-10 cells. (c-e) pancreaticacini were used to measure exocytosis in response to stimulation with the indicated CCK concentrations. (c) shows exocytosis during the first 5 min of stimulation, (d) shows the two phases, first rapid and second sustained phase, of exocytosis and (e) shows exocytosis at the indicated CCK concentrations measured during 30 min of stimulation. The results are averages of 3-4 experiments with each mouse line. denotes p<0.01 or better and # denotes p<0.05 or better.
aluRNA is localized to nucleoli and partially rescues α‐amanitin‐induced nucleolar dispersionCLSM images showing nucleolar co‐localization of NCL (immunofluorescence) with aluRNA (RNA FISH) but not with L1‐repeat RNA.Figure 3CLSM images showing nucleolar co‐localization of NCL (immunofluorescence) with aluRNA (RNA FISH) but not with L1‐repeat RNA. Cells were pre‐treated in situ with RNase A or an RNase inhibitor. Nuclei were counterstained with DAPI. The signal intensity of nucleolar aluRNA was two‐fold higher compared to nucleoplasmic signal (n = 92, P‐value 0.05, t‐test). Graphs representing the average number of nucleolar bodies after microinjection of in vitro transcribed RNA into HeLa cells that were pre‐treated with α‐amanitin (50 μg/ml) for 5 h or left untreated (control) (± 95% CI. **P‐value 0.01, n = 90, 87, 86, 83 or 86 cells, respectively). Representative CLSM images of propidium iodide‐stained RNA are shown on the right side.
The starvation‐dependent cycling of mammalian Atg9 (mAtg9) is blocked by inhibition of myosin II activity.Figure 8(A) Immunofluorescence analysis of GFP-mAtg9 and phospho‐myosin regulatory light chain (MRLC) in MCF7/GFP-mAtg9 cells. GFP-mAtg9 was enriched in the trans‐Golgi network (TGN) labelled with anti‐TGN46 antibody and phospho‐MRLC localized to cell peripheral in nutrient‐rich condition (DMEM). GFP-mAtg9 redistributed and co‐localized with phospho‐MRLC in starvation condition (Earle's balanced salt solution; EBSS). (B) mAtg9 interacted with non‐muscle myosin heavy chain‐IIA (NMHC‐IIA) under starvation conditions. 293T cells (right panel) or 293T cells stably infected with lentivirus expressing control (shLuc), UNC‐51‐like kinases (Ulk1) or zipper‐interacting protein kinase (ZIPK) shRNA (left panel) were transfected with V5‐tagged mAtg9 and GFP-tagged NMHC‐IIA. At 48 h after transfection, cells were incubated in either serum‐containing medium (DMEM) or EBSS for 2 h with or without the treatment of 50 μM ML‐7 and blebbistatin (Blebb). Cell lysates were immunoprecipitated with anti‐V5 antibody and immunoblotted with indicated antibodies. (C) MCF7/GFP-mAtg9 cells were stably infected with lentivirus expressing control (shLuc), ZIPK, or NMHC‐IIA shRNA. Under starved conditions, mAtg9 was redistributed from the TGN to a dispersed peripheral pool in control cells (shLuc). Starvation‐induced GFP-mAtg9 redistribution was blocked in ZIPK and NMHC‐IIA knockdown cells. The images were analysed by quantifying mAtg9 localization in cells for either a TGN‐enriched or dispersed pattern. Data are represented as mean±s.e. of 70 cells. *P0.05, **P0.01. Bar, 10 μm.
a) UVRAG interaction with C-Vps.figf1(a) UVRAG interaction with C-Vps. 293T cells were co-transfected with Flag-UVRAG and HA-Vps16, HA-Vps11, HA-Vps18, HA-Vps33 or HA-Vps39. Whole-cell lysates (WCLs) were used for immunoprecipitation (IP) with an anti-Flag antibody, followed by immunoblotting (IB) with an anti-HA antibody. 1% WCL was used as the input. (b) Interaction between endogenous UVRAG and C-Vps subunits. WCLs of 293T cells were used for IP with control serum (control) or an anti-UVRAG antibody, followed by IB with the indicated antibodies. The right panel shows endogenous protein expression. (c) Confocal microscopy analysis of the colocalization of endogenous UVRAG and Vps16 or Vps18 in HeLa cells. Insets highlight colocalization. (d) Confocal microscopy analysis of the colocalization of UVRAG with C-Vps subunits in HeLa cells transfected with Flag-UVRAG, and epitope-tagged C-Vps subunits as indicated. (e) Colocalization of UVRAG-C-Vps in early endosomes. HeLa cells were co-transfected with EGFP-Vps16 and Flag-UVRAG, stained with antibodies to LAMP1, LAMP2, EEA1 and Rab5, followed by confocal microscopy. All images are representative of at least three independent experiments. Scale bars, 5 μm. The raw data for a and b are shown in Supplementary Information, Fig. S6
Figure 2(A) Representative images of whole brains from wild-type (WT) and hFOXM1f/+-NesCre (hFOXM1-cKI) mice pups at P0. Scale bar,100μm. (B) Quantification of cortical width and length at P0 (WT n=7; hFOXM1-cKI n=7; two-tailed unpaired t-test, P= 0.0361for width, P= 0.2866 for length). (C) Quantification of brain/body weight at P0 (WT n=9; hFOXM1-cKI n=9; two-tailed unpaired t-test, P= 0.0004). (D) Quantification of cortex thickness at P0 (WT n=7; hFOXM1-cKI n=11; two-tailed unpaired t-test, P= 0.0002). (E) (E) Staining for Satb2 and Ctip2 in P0 WT and hFOXM1-cKI mice folding structure. Asterisks indicate gyrus and arrows indicate sulcus structures. A1, A2, B1and B2 (DAPI) are high-magnification images. Scale bars, 200μm(left); 100μm(right). (F) Quantification of GI of WT and hFOXM1-cKI mice pups at P0. The quantification of the GI was analyzed in pre-selected hFOXM1-cKI mice with folding. (n = 6 each group; two-tailed u unpaired t-test, P= 0.0010). (G）Staining for Satb2 and Ctip2 in P0 WT and hFOXM1-cKI mice. White dash lines represent the positive area. Scale bar,100μm. (H) Graph shows the number of Satb2+cells in 4×104µm2 at P0 (WT n=5; hFOXM1-cKI n=5; two-tailed unpaired t-test, P= 0.0086). (I) Graph shows the number of Ctip2 +cells in 4×104µm2 at P0 (WT n=5; hFOXM1-cKI n=5; two-tailed unpaired t-test, P= 0.0025). ( (J) Staining for DAPI in P3 WT and hFOXM1-cKI mice. Asterisks indicate gyrus and arrows indicate sulcus structures. Scale bars, 500μm . (K) Staining for Cux1 in P0 WT and hFOXM1-cKI mice. Scale bar,100μm. (I) Graph shows the number of Cux1+cells in 4×104µm2 at P0 (WT n=3; hFOXM1-cKI n=3; two-tailed unpaired t-test, P= 0.0451). D