EMBO/sd-character-level-ner huggingface dataset:
Figure 4A. Images of HeLa cells stably expressing tubulin-GFP (green) and transiently mRFP-CENP-B (magenta) treated with control siRNA (left images) and siRNA targeting PRC1 (right images). Enlargements of the boxed region, shown to the right of the image of the whole spindle, are focused on kinetochores and bridging fiber (top: merge; middle: GFP, bottom: scheme). B. Western blot showing PRC1 protein from unlabeled cells, tubulin-GFP HeLa cell line and PRC1-GFP HeLa cell line. Detailed measurements are in Expanded View 4. C. Mean ratio of signal intensities of the bridging fiber (Ib, measured at the position of a blue line as in scheme) and sum of the bridging and k-fiber (Ibk, measured at the position of the orange line as in scheme), measured in control cells (grey bar) and PRC1 siRNA treated cells (black bar) (p=0.0003).D. Mean interkinetochore distance (dk, see scheme) measured in control cells (grey bar) and PRC1 siRNA treated cells (black bar) (p=0.0001). E. Images of individual spindles of HeLa cells expressing tubulin-GFP and immunostained for PRC1 obtained from control cells (top) and PRC1 siRNA treated cells (bottom). All cells were imaged with the same imaging parameters. Only AlexaFluor555 channel is shown. F. Mean signal intensity I of the immunostained PRC1 in control cells (grey bar) and PRC1 siRNA treated cells (black bar) in HeLa cells expressing tubulin-GFP and immunostained for PRC1 (p=0.0001). G. Images of HeLa cells stably expressing PRC1-GFP treated with control (left image) and siRNA targeting PRC1 (right image) immunostained for PRC1 (magenta). Graph showing the length of the immunostained PRC1 signal, LPRC1 (p=0.5167), signal intensity, I (p=0.0003) and Icross (p=0.0001) in siRNA targeting PRC1 in comparison to control HeLa cells expressing PRC1-GFP and immunostained for PRC1.H. Images of HeLa cells stably expressing PRC1-GFP (green) treated with control (left image) and siRNA targeting PRC1 (right image) immunostained for PRC1. Graph showing the length of the PRC1-GFP signal, LPRC1 (p=0.3407), signal intensity, I (p=0.0001), and Icross (p=0.0001) in siRNA targeting PRC1 in comparison to control HeLa cells expressing PRC1-GFP and immunostained for PRC1.
DIA1(R1204X) induces elongated and thick microvilli in HeLa cells.Figure 3Various GFP-tagged DIA1 plasmids were transfected into HeLa cells, and fixed 24 h after transfection. Fixed cells were stained with Alexa568-conjugated phalloidin and DAPI (A, B: representative of 5 experiments).(A) Stress fiber formation was observed with a confocal laser microscope. Note the induction of stress fibers by GFP-DIA1(M1190D) (asterisks). Arrow and arrowheads indicate a plasma membrane (PM) localization of GFP-DIA1(M1190D) and a dot-like localization of GFP-DIA1(R1204X) adjacent to the PM, respectively. Inset in the top panel of DIA1 (R1204X) shows the magnified image of the region indicated by rectangle. Scale bars: 10 μm.. Various GFP-tagged DIA1 plasmids were transfected into HeLa cells, and fixed 24 h after transfection. Fixed cells were stained with Alexa568-conjugated phalloidin and DAPI (A, B: representative of 5 experiments).(B) Microvilli with or without GFP-DIA1(R1204X) expression were observed under a confocal laser microscope. Note elongated and thick microvilli (arrowheads) in GFP-DIA1(R1204X)- transfected cells, but not in non-transfected cells (circles). 3D movies are available in Movie EV1 (R1204X), Movie EV2 (WT), and Movie EV3 (ttaa). Scale bar: 10 μm.
Transcriptomic profiles of Black versus White HKCs are characterized by an OXPHOS-related gene signature.Figure 3(A) Top bar: gradient of population admixture of the indicated HKC strains of Black versus White individuals, on the basis of the SNP genotype analysis shown in Figure 1B, with the simplest assumption of two ancestries (K = 2). Gradient ranges from black to white colors for Black and White individually with no population admixture, respectively. Individuals with increasing proportions of White population admixture are represented with degrading shades of grey. Bottom: Heatmap of differentially expressed genes (characterized transcripts) in HKC strains from Black versus White individuals ordered according to genome admixture calculations. 164 genes with significantly different expression levels between the two groups (log2(Foldchange)|>0.58 and P value <0.05) were identified (Dataset EV1). Modified Z scores of the individual genes, as median-centered log2 Intensity values divided by standard deviation, are shown by color gradient variations. Unsupervised hierarchical clustering of genes was based on their modified Z scores, using Manhattan distance as distance metric and complete linkage method (Shirkhorshidi et al., 2015). 4 main groups of co-expressed genes were identified (purple triangles). (B) Gene Ontology (GO) analysis of the differentially expressed genes identified in the previous panel. Shown is a list of process networks with statistical significance of enrichment in Black HKCs, total or with < 20% genomic admixture (upper and lower panels, respectively). The complete list is provided in Dataset EV1.Fisher's Exact p-values are reported. (C) Gene set enrichment analysis of the global expression profiles of the Black versus White HKC strains (GSE156011) using 50 HALLMARK signatures (https://www.gsea-msigdb.org/gsea/msigdb/collections.jsp#H), revealing a significant association with the OXPHOS signature. Genes are ranked by signal-to-noise ratio based on their differential expression in Black versus White HKC strains. Position of genes in each gene set is indicated by black vertical bars and the enrichment score (ES) by the green line. Normalized Enrichment Score (NES), nominal p-value and FRD q-value, based on the permutation-generated null distribution, are as shown.
Single cell analysis of the public kidney data of three donors.Figure 1.UMAP plot revealing the cell type annotations of three samples. B) The cellular composition of three healthy kidney samples. Dotplot exhibition of canonical markers used for cell type classification. The gene expression patterns of ACE2 of all detected cell types in kidney.
Figure 7A, B Quantitative real time (qRT)-PCR analysis of MdCAX3 or MdCXIP1 in the silencing MdCAX3 or MdCXIP1 Mx roots under control (Hoagland nutrient solution) and excess Zn (300 μM Zn) conditions for 3 weeks. Values are means ± SD, n = 3 (biological replicates). Asterisks indicate statistically significant differences (***, P < 0.001, Student's t-test), ns = no significant difference. C Shoot phenotypes of Mx under control (Hoagland nutrient solution) and excess Zn (300 μM Zn) conditions for 3 weeks. Scale Bar: 1 cm. D Perls stain and Zinquin ethyl ester assays of Mx under control (Hoagland nutrient solution) and excess Zn (300 μM Zn) conditions for 3 weeks. Leaf Scale Bar: 0.5cm, Root Scale Bar: 0.5 mm.
Figure 4A The five most strongly induced genes (right column) upon TGFβ treatment of TA organoid cultures are higher expressed in SSAs when compared to TAs, whereas the five most strongly reduced genes (left column) follow the opposite pattern (n = 12 for SSA and n = 15 for TA. The horizontal lines represent the mean, the error bars display the SD. P-values are based on unpaired two-tailed Student's t-tests.). B Hierarchical clustering of 15 TA and 12 SSA samples using a TGFβ response signature derived from TGFβ-treated TA organoids (log2FC > 3). Expression values were mean-centered (gene-wise) and cosine similarity was used as the distance measure. C Genes induced upon TGFβ treatment of TA organoids, that are differentially expressed between TA and SSA samples, display significantly higher expression levels in CMS4-SSAs compared with CMS1-SSAs (n = 8 for CMS1-SSAs and n = 4 for CMS4-SSAs. The horizontal lines represent the mean, the error bars display the SD. P-values are based on unpaired two-tailed Student's t-tests.). D Gene expression-based classification reveals that SSAs could progress to CMS1 and CMS4 tumors. The data presented in this manuscript suggest that high activity of the TGFβ signaling pathway directs these precursor lesions to the CMS4 of CRC.
Figure 2(A-C) Whole-cell recordings of spontaneous epileptiform activity from cultured hippocampal neurons. A, representative traces showing the neuronal activity under the indicated treatments. Ctrl: extracellular solution. Cyclo: cyclopamine. B, the expanded view of a single burst (arrow) from A; C, statistics of the percentage of neurons showing epileptiform activity (left, n=6-7) and the burst frequency (right, n=18-19) shown in A. Cells were incubated in the presence of Cyclo in extracellular solution for 30 min, then transferred to 0Mg plus Cyclo (0Mg+Cyclo) and incubated for another 30 min. (D,E) The effects of Sant-1, robotnikinin (Robot) or 5E1 on the percentage of neurons showing epileptiform activity (D, n=6-10) and the burst frequency (E, n=15-22). (F-H) Extracellular recordings of spontaneous epileptiform activity from CA1 stratum pyramidal of hippocampal slices. F, representative traces under the indicated treatments. Ctrl: artificial cerebrospinal fluid (aCSF). (F-H) Extracellular recordings of spontaneous epileptiform activity from CA1 stratum pyramidal of hippocampal slices. F, representative traces under the indicated treatments. Ctrl: artificial cerebrospinal fluid (aCSF). G, the expanded view of a single burst (arrow) in F. Slices were incubated in the presence of vehicle or Cyclo in aCSF for 30 min, then transferred to picrotoxin in aCSF (Pic) or with Cyclo (Pic+Cyclo) and recorded for 30 min. H, statistics of the spontaneous burst frequency of F from 10 slices (7 rats). (I,J) Left are representative traces showing the D-CPP-sensitive currents recorded from cultured hippocampal neurons (I) or slices (J) incubated in 0Mg or Pic, respectively, with or without Cyclo. Black lines: application of D-CPP. Right are statistics of D-CPP-sensitive current density from 18-26 cells (I) or 16-18 slices (15 rats) (J). Unless stated, 10 µM cyclopamine was used. Data were mean + SEM. *p<0.05,**<0.01,***p<0.001 vs. Ctrl; #p<0.05,##<0.01,###p<0.001 vs. 0Mg or Pic; &amp;amp;p<0.05 vs. 0Mg + IgG. C-E used one-way ANOVA. H-J used student's t test.
Loss of FBXO7 in TH+ cells leads to reduced striatal dopamine.Figure 8A. Images of representative 12 month-old TH-Cre and TH-Cre;fl/fl midbrains immunostained with the TH antibody (black) and Nissl stain (purple). Scale bar = 500 µm. Scale bar of inset 10 µm. B. and C. Stereological analysis of ratio of TH+Nissl+ cells/Nissl+ cells in the substantia nigra (A) from 2 month-old female (B) and 12 month-old male (C) TH-Cre and TH-Cre;fl/fl mice. Three animals from each genotype were analyzed (unpaired t-test, mean +s.e.m.). D. E. F. and G. Striatal tissue from P18 TH-Cre and TH-Cre;fl/fl mice was subjected to HPLC analyses to determine dopamine (D), 3,4-dihydroxyphenylacetic acid (DOPAC) (E) and homovanillic acid (HVA) (F) concentrations along with the metabolite ratio (G). N=7 and 9 (2 months) and n=15 and 12 (12 months), (unpaired t-test, * p<0.05, *** p<0.001, mean +s.e.m.) H. Images of representative TH-Cre and TH-Cre;fl/fl striatal sections immunostained with the TH antibody. Scale bar = 1 mm. I. Immunohistochemistry of midbrain section TH-Cre and TH-Cre;fl/fl mice using the TH and GFAP antibody. Quantitative measurements of GFAP stainings from four animals per genotype (unpaired t-test, ***p<0.001, mean + s.e.m.).
Figure 5The number of overlapped peaks (1186) and genes (1085) in two ChIP-seq experiments. Statistical differences were determined using Fisher exact test. Correlation of read density among the 1186 peaks between two ChIP-seq experiments. The enriched DNA motif is identified by the motif-discovery algorithm MEME in AP2-O3 binding sites. EMSA using the recombinant GST-fused AP2 domain of AP2-O3 and a synthesized DNA probe containing three repeats of predicted motif. GST was used as a negative control. An arrowhead indicates the shifted band. The number (319) of overlapping genes between AP2-O3-bound genes (1085) in ChIP-seq and upregulated genes (1141) in RNA-seq. Statistical differences were determined using Fisher exact test. Gene ontology enrichment analysis of the 319 genes indicates male-specific or preferential biological processes. Hypergeometric test was applied. Mapped views showing the RNA-seq and ChIP-seq results of four male genes in DNA replication and DNA repair pathways. ChIP-qPCR validation of the binding between AP2-O3 and the upstream region of four male genes The regions detected via ChIP-qPCR are indicated by pink line (in the peak) and purple line (out of the peak). mean±SEM from three independent experiments.
Figure 5Heatmap of differentially expressed transcripts in RNA-seq data from GBM#P3 cells treated with DMSO or 20 μM 24OHC for 72 h. Gene expression data was mean z-transformed for display, and colored red for high expression and blue for low expression. GO and KEGG enrichment analysis of differentially expressed genes based on RNA-seq data with adjusted-P value. GSEA plot showing normalized enrichment scores (NES) for cholesterol homeostasis, positive regulation of cholesterol efflux, SREBP targets and stem cell proliferation signatures using RNA-seq data from 24OHC and DMSO treated cells. Volcano plot highlighting up-regulated (red) and down-regulated (blue) genes based on RNA-seq data from GBM#P3 cells after 24OHC treatment. LXR targets, SREBP targets and stemness transcription factors are specifically included. qRT-PCR analysis of 24OHC-regulated genes in GBM#P3 cells after treatment with 20 μM 24OHC for 72 h. GAPDH was used as an internal control. Data are shown as the mean ± SEM (n = 3). Cell viability assay performed on GBM#P3 cells after 3 days of treatment with DMSO or 5 -10 μM LXR-623 in the presence or absence of 10 μM 24OHC. Data are shown as the mean ± SEM (n = 3). Western blot analysis of cholesterol metabolism-related proteins in LN229 or GBM#P3 after treatment with 24OHC (0 - 20 μM). Growth curves for LN229 cells transfected with control vector or SREBP1 plasmid and treated with 24OHC (0 - 40 μM) as assessed by a trypan blue test. Data are shown as the mean ± SEM (n = 3). Colony forming ability of GBM cell lines treated with DMSO or 20 μM fatostatin for 14 days. Representative images of filipin staining in LN229 (up) or LN18 (down) cells treated with different concentration of fatostatin (0 - 20 μM). Scale bar = 30 μm.
Figure 3A Western blot of lysed transgenic nematodes that express the UbG76V-GFP and mRFP reporters in the hypodermis at the L4+48h stage, probed with antibodies recognizing GFP or actin as a loading control. The position of molecular weight markers is shown on the left of each blot. The position of UbG76V-GFP protein, as well as UbG76V-GFP with the indicated number of additional ubiquitin moieties, based on molecular weight, is indicated to the right of each blot. Twenty animals were loaded per lane for each indicated genotype. B A separate SDS-PAGE and Western blot of the same lysates from (A), probed with antibodies recognizing ubiquitin or actin as a loading control. The position of molecular weight markers is shown on the left of each blot. The position of UbG76V-GFP protein, as well as UbG76V-GFP with the indicated number of additional ubiquitin moieties, based on molecular weight, is indicated to the right of each blot. Endogenous poly-ubiquitinated proteins are also detected in the 80-200 kDa range, as indicated by the bracket. Twenty animals were loaded per lane for each indicated genotype. C Quantified UbG76V-GFP protein levels relative to actin control for each indicated lane for the blot in (A). ****P<0.0001, ANOVA with Dunnett's posthoc comparison to wild type. N=3 trials. Error bars indicate SEM. D Quantified UbG76V-GFP protein levels relative to actin control for each indicated lane for the blot in (B). ****P<0.0001, ANOVA with Dunnett's posthoc comparison to wild type. N=3 trials. Error bars indicate SEM. E Quantified endogenous poly-ubiquitinated protein levels relative to actin control for each indicated lane for the blot in (B). ****P<0.0001, ANOVA with Dunnett's posthoc comparison to wild type. N=3 trials. Error bars indicate SEM. F Quantified fluorescence of UbG76V-GFP normalized to mRFP from the same lysates. *P<0.01, ***P<0.001, ANOVA with Dunnett's posthoc comparison to wild type. N=3 trials. Error bars indicate SEM. G Quantified epoxomicin-sensitive proteasome activity (as measured through the turnover of fluorescent chymotrypsin substrate) from the same lysates. **P<0.01, ANOVA with Dunnett's posthoc comparison to the wild-type control, with N=3 trials. Error bars indicate SEM.
ACS2 Depletion Ameliorates Age-Associated Autophagy in Yeast (A) Scheme of known major acetyl-CoA (AcCoA)-producing enzymes (bold characters) modulated in this study.Figure 1(A) Scheme of known major acetyl-CoA (AcCoA)-producing enzymes (bold characters) modulated in this study. (B) Representative immunoblot of GFP-Atg8p expressing yeast. Wild-type cells (tet-WT) were compared to strains carrying a doxycycline-repressible tet-O7 promoter controlling ACS2 transcription (tet-ACS2). Cells were grown in SC 2% glucose medium for 24 hr (day 1 of aging) in the presence (+Doxy) or absence (−Doxy) of 1 ng/ml doxycycline to induce knockdown of ACS2 (see also Figure S1). (C) Fluorescence microscopy of GFP-Atg8p expressing (under control of its natural pATG8 promoter) wild-type (tet-WT) and ACS2 knockdown (tet-ACS) cells grown in the presence of 1 ng/ml doxycycline (as shown in A) and chronologically aged for 3 days. Propidium iodide (PI) counterstaining served to visualize dead cells. Scale bars represent 5 μm.(D) Quantification of cells depicted in (C) (day 3) and of young (day 1) cells with 150-300 counts (blinded) for each replicate. Autophagic cells were defined as cells with clear vacuolar GFP fluorescence. Data represent means ± SEM (n = 4). ∗∗p < 0.01.(E) Representative immunoblot analysis of day 1 (young controls) and day 3 (aged) cells shown in (C) and (D) using anti-GFP and anti-GAPDH (loading control) antibodies to detect "free-GFP" indicative of autophagic flux. (F-H) Representative micrographs (F), respective quantification (G), and immunoblot analysis (H) of wild-type (WT) and ACS1-deleted (Δacs1) yeast aged to day 3 compared to young (day 1) cells expressing GFP-Atg8p chimera as in (C)-(E). Data represent means ± SEM (n = 4).
Figure 3Scatter plots showing the correlation between FOXA2 ChIP-seq biological duplicates in CFPAC1 (top panel) and PANC1 cells (bottom panel). Pearson's correlation (r) between samples is indicated. Scatter plot showing the correlation between FOXA2 ChIP-seq peaks in CFPAC1 and PANC1 cells. Green and blue dots indicate FOXA2 peaks that are significantly (FDR < 0.001) different between the two cell types and with log2 fold enrichment ≥ 2 as determined by DiffBind. All other peaks are indicated as grey dots. The boxplots on the right show the intensity of CFPAC1-specific, PANC1-specific and other FOXA2 peaks. Central values represent the median, the bars the 25th and 75th percentile and the vertical lines the lower and upper whiskers. Distribution of the positions of the FOXA motif relative to the summit of different sets of FOXA2 peaks. The PWM used to perform CentriMo analysis and the E-value for each dataset are shown. Representative FOXA2 ChIP-seq snapshot showing FOXA2 peaks classified as CFPAC1-specific, shared or PANC1-specific. GO terms retrieved by GREAT analysis associated with distal FOXA2 peaks specific of CFPAC1 (left) or PANC1 (right). Terms are ranked by the negative log10 p-value from binomial correct test.
Figure 7Representative images (A) or traces (B-D & E-G) of changes in fluorescence of synaptophysin-pHluorin upon stimulation of neurons (scale bar in panel (A) = 2 µm). Plot of change in fluorescence from baseline to peak fluorescence for Munc18-1 G544D (H) Data are means ± SEM (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by one-way one-way ANOVA and Dunnett's multiple comparison test; n = 7-23; exact n and p values are shown in Appendix Table S1). Note, that compound 10 is fluorescent at a similar wavelength than pHluorin, so experiments at concentrations higher than 1 µM were not possible. Plot of change in fluorescence from baseline to peak fluorescence for Munc18-1 R406H (I). Data are means ± SEM (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by one-way one-way ANOVA and Dunnett's multiple comparison test; n = 7-23; exact n and p values are shown in Appendix Table S1). Note, that compound 10 is fluorescent at a similar wavelength than pHluorin, so experiments at concentrations higher than 1 µM were not possible.
Defective meiotic progression in the first wave of spermatogenesis in Tex11 KI/KO males.Figure 3Reduced testis weight in juvenile but not adult Tex11 KI; −/Y males. The time frames of the first wave and adult waves of spermatogenesis are indicated. Increased chromosomal asynapsis in spermatocytes and reduced levels of TEX11 protein in the testes of 25-day-old Tex11 KI/KO males. Chromosomal synapsis defects were assessed by SYCP1 and SYCP2 immunostaining of spread nuclei from 100 pachytene spermatocytes per male; for each genotype, three males were analyzed. Pachytene spermatocytes from 25-day-old Tex11 KI/KO males contain significantly fewer MLH1 foci compared to wild-type spermatocytes. Values are shown as average ± standard deviation. Error bar is standard deviation.
a, shCtrl or shXbp1 MODE-K cells were co-silenced for Atg16l1 (siAtg16l1) or with scrambled siRNA (siCtrl), and analysed by flow cytometry for annexin V and propidium iodide (PI; n = 3; one-way ANOVA with post-hoc Bonferroni; mean ± s.f3a, shCtrl or shXbp1 MODE-K cells were co-silenced for Atg16l1 (siAtg16l1) or with scrambled siRNA (siCtrl), and analysed by flow cytometry for annexin V and propidium iodide (PI; n = 3; one-way ANOVA with post-hoc Bonferroni; mean ± s.e.m.). b, Immunoblot of primary intestinal epithelial cell scrapings (n = 3). c, Immunoblot of cytoplasmic extracts from shCtrl or shXbp1 MODE-K cells after TNF stimulation. d, TUNEL+ intestinal epithelial cells (IECs) per 100 crypts after BAY11-7082 or vehicle treatment (n = 3/4/4; one-way ANOVA with post-hoc Holm's-corrected unpaired Student's t-test; mean ± s.e.m.). e, Enteritis histology score of mice treated with BAY11-7082 or vehicle (n = 10/10/9; median shown; Kruskal-Wallis with post-hoc Holm's-corrected Mann-Whitney U-test). f, Immunoblot of intestinal epithelial cell scrapings for (p-)IRE1α; after IRE1α; immunoprecipitation (IP). beta-actin, loading control of whole lysates. g, Immunoblot of primary intestinal epithelial cell scrapings (n = 4). h, i, Enteritis histology score of indicated genotypes (h, n = 15/16/14/15; i, n = 5/10/12; median shown; Kruskal-Wallis with post-hoc Holm's-corrected Mann-Whitney U-test). j, Enteritis histology score of specific pathogen-free (SPF) and germ free (GF) housed mice (n = 10/9/7/7; median shown; Kruskal-Wallis with post-hoc Holm's-corrected Mann-Whitney U-test). k, Representative images of p-IκBα immunoreactivity under conditions as in j (n = 4). Scale bars, 20 µm. Results represent four (f), three (c) or two (a, b) independent experiments. *P  0.05, **P  0.01, ***P  0.001.
Placensin stimulation of cAMP production, PKA activity, and glucose release in hepatocytes and promotion of glucose secretion in vivo.Figure 3A Prokaryotic cell-derived placensin stimulated cAMP production (at 10 min.), and PKA activity (at 30 min.) by primary mouse hepatocytes and hepatocyte-derived AML12 cells (biological replicates, n=5). B Placensin stimulation of glucose secretion (at 5h) and key gluconeogenesis gene transcripts (at 1h) in both cell types (biological replicates, n=4). Transcript levels for PEPCK and G6Pase were determined using quantitative RT-PCR. Treatment with glucagon (10 nM) served as positive controls. C Liver from adult female mice was perfused in situ with placensin (1.0 ug/pulse) or PBS before measurement of hepatic glucose output from inferior vena cava (biological replicates, n=3). D Adult female mice were injected intraperitoneally with placensin (30 ug/injection) with blood sampling at different time points for glucose levels (biological replicates, n=5).