EMBO/sd-character-level-ner huggingface dataset:
CRISPR/Cas9 based PYCR1 knockout increases glutamine flux to the TCA cycle.Figure 5A) Seahorse experiment measuring oxygen consumption rate (OCR) in MCF7 cells upon addition of oligomycin, FCCP and a mixture of antimycin and rotenone. Arrows indicate the points of addition of each inhibitor. Data are presented as mean ± SD of one representative experiment (n = 4 wells). Similar results were obtained in three biological experiments. Samples were compared using Kruskal Wallis test followed by Dunnett's test for multiple pairwise comparisons. Corrected p values were indicated as follows ** p < 0.01 and *** p < 0.001. Shapiro Wilk test was used to check data normality and Bartlett test was used to examine homogeneity of variances. B) Bar plot indicates average basal OCR and spare respiratory capacity in seahorse measurements. Data are presented as mean ± SD of one representative experiment (n = 4 wells). Samples were compared using Kruskal Wallis test followed by Dunnett's test for multiple pairwise comparisons. Corrected p values are indicated as follows *p < 0.05 and *** p < 0.001. Shapiro Wilk test was used to check data normality and Bartlett test was used to examine homogeneity of variances. C) 13C5 -Glutamine derived carbon labeling patterns of TCA cycle intermediates fumarate, malate and citrate in MCF7 cells. Data are presented as mean ± SD of triplicate samples. Isotopologues are represented as M+n where M indicates the mass and n equals number of 13C incorporated D) Seahorse experiment measuring extracellular acidification rate (ECAR) in MCF7 cells upon addition of glucose, oligomycin and 2-deoxyglucose (2DG). Arrows indicate the points of addition of each inhibitor. Data are presented as mean ± SD of one representative experiment (n = 4 wells). Samples were compared using Kruskal Wallis test followed by Dunnett's test for multiple pairwise comparisons. Corrected p values are indicated as follows ** p < 0.01. Shapiro Wilk test was used to check data normality and Bartlett test was used to examine homogeneity of variances. E) Bar plot indicates average glycolytic capacity and glycolytic reserve in seahorse measurements. Data are presented as mean ± SD of one representative experiment (n = 4 wells). Similar results were obtained in three biological experiments.Samples were compared using Kruskal Wallis test followed by Dunnett's test for multiple pairwise comparisons. Corrected p values are indicated as follows ** p < 0.01 and *** p < 0.001. Shapiro Wilk test was used to check data normality and Bartlett test was used to examine homogeneity of variances. F) Normalized lactate concentration upon PYCR1 KO. Data represents mean ± SE of three biological experiments. Samples are compared using Kruskal Wallis test followed by Dunnett's test for multiple pairwise comparisons. Corrected p values are indicated as follows *p < 0.05. Shapiro Wilk test was used to check data normality and Bartlett test was used to examine homogeneity of variances.
Figure 5(A) RPE1 cells were transfected with siRNAs as indicated, followed by 24 h incubation in serum-starved media. Cells were then and subjected to fractionation, and Western blot for IFT20, the Golgi marker GM130, membrane marker UBXD8, and nuclear marker CREB. (B) Efficiency of IFT20 knockdown by western blot. (C) Cells were transfected with siRNAs as indicated, followed by incubation in serum-starved media for 24 h, and immunostained for ARL13B (red). Scale bar, 10 μm. The bar graph represents the quantification of the percentage of ciliated cells. Data represent mean ± SD (n=3 experiments), 250 cells were scored per condition per experiment, *P < 0.05, Student's t-test. (D) Cells were transfected and immunostained for Rab8 and γ-tubulin, followed by quantification of the percentage of cells with Rab8 localized to the centriole. Data represent average (n=2 experiments). (E) Cells were transfected with siRNAs as indicated, followed by transfection with Flag-IFT20, incubation in serum-starvation media for 12 h, and immunostained for ARL13B. Representative fluorescent images of Flag-IFT20 (green), ARL13B (red), and DAPI (blue) are shown. Scale bar, 10 μm. (F) Quantification of the percentage of ciliated cells with both the Flag-IFT20 and ARL13B localized in the cilium. Data represent mean ± SD (n=3 experiments), 150 Flag positive cells were scored per condition per experiment, *P < 0.05, Student's t-test. (G) Cells were transfected with siRNAs as indicated, followed by further transfection with CA-Rab8, incubation in serum-starvation media for 12 h, and immunostained for acetylated-tubulin. Representative fluorescent images of GFP-Rab8 Q67L (green), acetylated-tubulin (red), and DNA (blue) are shown. Scale bar, 10 μm. (H) Quantification of the percentage of GFP -positive ciliated cells (only those cilia having both GFP-Rab8 and acetylated-tubulin on cilium were considered for quantification). Data represent mean ± SD (n = 3 experiments), 200 GFP positive cells were scored per condition per experiment, *P < 0.05, Student's t-test.
Figure 6Accumulations of MITOL and IRE1α in the MAM fraction. Each organelle fraction was isolated from MEFs by percoll density-gradient centrifugation, followed by immunoblotting with indicated antibodies. mito: pure mitochondria, micro: microsome, cyto: pure cytosol. High solubility of MITOL and IRE1α in the low dose digitonin. Raft-like structure including MAM was solubilized from the crude mitochondrial fraction of MEFs with indicated concentration of digitonin. S: supernatant, P: pellet. IRE1α regulation by MITOL occurred in the MAM-rich mitochondrial fraction. Crude mitochondrial fraction containing MAM (Mito plus MAM) and the cytosolic fraction containing ER (Cyto plus ER) were isolated from HEK293 cells transfected with indicated vector and subsequently immunoprecipitation assay was performed to detect the interaction between MITOL and IRE1α IRE1α regulation by MITOL occurred in the MAM-rich mitochondrial fraction. Crude mitochondrial fraction containing MAM (Mito plus MAM) and the cytosolic fraction containing ER (Cyto plus ER) were isolated from HEK293 cells transfected with indicated vector and subsequently immunoprecipitation assay was performed to detect the level of IRE1α ubiquitylation (D). Knockdown of PACS2 impaired the interaction of MITOL with IRE1α. HEK293 cells were transfected with either scramble RNA or siPACS2 and indicated vectors for 24 hours prior to immunoprecipitation assay. Knockdown of PACS2 inhibited IRE1α ubiquitylation by MITOL. HEK293 cells were transfected with either scramble RNA, siPACS2 and indicated vectors 24 hours prior to immunoprecipitation assay. Knockdown of Mfn2 inhibited IRE1α ubiquitylation by MITOL. HEK293 cells were transfected with either scramble RNA, siMfn2 and indicated vectors 24 hours prior to immunoprecipitation assay.
Figure 4Passaging capacity analysis of the CLESCs derived from P7 WT and Prom1 KO mice. Note the passages of cells derived from WT animals are still ongoing while those from Prom1 KO mice were arrested at the first passage. Arrows indicated cells were still under passaging while blunt bars represent cells could not be passaged and stopped at the indicated passage. Clonogenicity assay for colony number (B) and size (C) after the first the passage of CLESCs derived from WT vs. Prom1 KO mice in the absence or presence of SHH (100ng/ml). Cells were seeded in triplicate on 24-well plates as 5,000 cell/per well. Sphenoid analyses. For these assays, epithelial and mesenchymal cells were mixed at ratio of 5,000:5,000 per well in triplicates on 96-well plates and grown for 3 days on Matrigel. Representative micrographs of entire well under indicated conditions are shown. Quantification of sphere diameter on samples presented in (D).
Elevated GABA inhibits mitophagy in mammalian cells A,B  Example images of Parkin-expressing HeLa cells analyzed using a tandem fluorochrome protein (mito-RFP-GFP) mitophagy assay under (A) control conditions or (B) displaying mitophagy depicted by the red mitochondrial structures localized to lysosomes.Figure 7A,B Example images of Parkin‐expressing HeLa cells analyzed using a tandem fluorochrome protein (mito‐RFP‐GFP) mitophagy assay under (A) control conditions or (B) displaying mitophagy depicted by the red mitochondrial structures localized to lysosomes. Bar, 10 μm. C Percentage of cells displaying mitophagy + s.d., **P < 0.01 using an unpaired two‐tailed t‐test, n > 80.
Expression analysis of genes related to epigenetic regulation.Figure 3(a) Clustered expression heatmap. (b) Expression of individual significantly up-regulated genes marked with # in (a). Asterisks indicate timepoints of significantly different expression (Wald test, Benjamini & Hochberg corrected, q<0.05) between stem and non-stem cells for each time point (N = 2).+ = stem cells; - = non-stem cells, E = nuclei from embryos, D7/14/35 = nuclei from 7/14/35 day-old plants, S14 = nuclei from 14 d-old above-ground seedlings.
Overexpression of inactive GAPDH rescues polyglutamine-induced blunted mitophagy and recovers mitochondrial function.Figure 5Representative Western blot showing levels of mitochondrial proteins, as indicated by the presence of aconitase, VDAC, and Tom20, in the total lysates of PC12 cells with Q23 or Q74 before (represented as NT: no treatment) and after overexpression of inactive GAPDH (represented as G). n = 3. *P < 0.035; **P = 0.001; #P = 0.001. Representative Western blot showing levels of mitochondrial proteins in the total lysates of mice striatal cells with Q7 or Q111 before (NT) and after overexpression of inactive GAPDH (G). n = 2. *P = 0.006; **P = 0.007; #P = 0.001. Determination of mitochondrial ROS production in different HD models. Results are shown as percent of control. n = 3. *P = 0.008; #P = 0.054; **P = 0.03. Determination of mitochondrial membrane potential using JC-1 in different HD models and control cells (MMP, ΔΨm). The difference in membrane potential between NT (no inactive GAPDH overexpression) and G (inactive GAPDH overexpression) in cells with expanded polyglutamine repeats (PC12 cells with Q74 and HD patient-derived fibroblasts) was not statistically significant. n = 3. ATP levels in normal and HD patient-derived fibroblasts. The difference in ATP levels between NT (no inactive GAPDH overexpression) and G (inactive GAPDH overexpression) in HD patient-derived fibroblasts was not statistically significant. n = 2. Cell viability determined by colorimetric assays in different HD models and control cells. n = 3. *P = 0.02; **P = 0.02; ##P = 0.01. Immunofluorescence of normal and HD patient-derived fibroblasts, with and without overexpression of inactive GAPDH, stained for late endosome with anti-Rab7 antibody and mitochondria with anti-Tom20 antibody. Images were acquired at 63× magnification, and brightness and contrast of all images were adjusted by 30%. Correlation coefficient (juvenile): **P = 0.007; *#P = 0.005. Mitochondrial interconnectivity (juvenile): ##P = 0.001; ##*P = 0.02. Correlation coefficient (adult): §§P = 0.001; *§P = 0.005. Mitochondrial interconnectivity (adult): ≠≠P = 0.043; ≠≠*P = 0.015.
Figure 3A Levels of pro- and anti-inflammatory cytokines in PBMC culture supernatants with CVF, CLys and CSN (10µg/ml) after 72h determined using a LUMINEX-based technology. B Composition and levels of bioactive lipid mediators (LMs), including prostaglandins, leukotrienes, lipoxins, thromboxanes, and their precursors in culture with CVF, CLys and CSN quantified by LC-MS/MS-based profiling. Heat maps show relative amounts of LMs detected by LC-MS/MS in supernatants. Asterisks show statistical differences analyzed using two-way ANOVA followed by Turkey's multiple comparison test. C Dynamic of PGE2 release correlated with Treg induction by CLys (upper panel) and CSN (lower panel) using a Pearson correlation. D Treg induction by CLys or CSN following PBMC pre-incubation with 10µM mPGES1 inhibitor 934 (or DMSO as a control). E Induction of Tregs with PGE2 analog (DINP) (5µM) and/or recombinant human IL-10 (rhIL-10) (10ng/ml) as compared to CLys.
Cx43 interacts with hnRNPA2B1 and hnRNPQ.Figure 3A. Cx43 was immunoprecipitated (IP) from HEK293Cx43+ cells, after which interaction with hnRNPA2B1 and hnRNPQ was analyzed by WB. B. Co-localization between Cx43 (green) and hnRNPA2B1 or hnRNPQ (red) in C33a cells was analyzed by confocal microscopy. Scale bars 10 μm. Arrowheads in the inset images highlight co-localization between Cx43 and hnRNPs.
Figure 1Isosurface of the subtomogram average (in pink) with an applied C5 symmetry and 0.69 nm tomographic slices (A-E) at the indicated heights. Scale bars 10 nm. The average is shown in side view, whereas the slices represent perpendicular slices. The 5 pairs of pillars formed a narrow central channel and separated into 10 arches towards the base (base not visible at the used threshold). The division of the structure into subparts was adapted from (Durand et al., 2015). Subvolumes were extracted from cryotomograms of ghost cells and FIB-milled intact E. coli BL21 cells heterologously expressing TssJLM Slice (0.69 nm) through the non-symmetrized average. Note that the C5 symmetry was visible
Fig.Figure 5(A, B) Immunoprecipitation with various lipid-coated beads using HeLa NOD1-GFP (A) and NOD2-GFP cells (B). Cell lysates of HeLa NOD1 or HeLa NOD2 cells were incubated with lipid-coated beads at room temperature for 2 h. (C) Immunoprecipitation with S1P-coated beads using THP-1 lysates. (D) Dual-color dSTORM imaging of HeLa NOD1 or NOD2 upon S1P stimulation. After stimulation with S1P-TAMRA (20 µM) for 2 h, HeLa NOD1 or NOD2 cells were fixed, permeabilized and stained with anti-GFP antibody and Alex Fluor 647-conjugated secondary antibody. Images were reconstructed from 10,000 raw frames. Scale bar: 200 nm. (E, F) Single particle tracking of NOD1-GFP (E) or NOD2-GFP (F) with S1P-TAMRA. After doxycycline induction overnight, HeLa NOD1 or NOD2 cells were stimulated with S1P-TAMRA (20 µM) for 1 h together with digitonin. Scale bar: 5 µm.
Figure 1Distribution of 25-35 mer reads mapping to the mouse genome. Proportion of 30-35 nt genomic reads matching mouse tRNA sequences. The sequencing was done in duplicates. Error bars represent SEM, significance was calculated using unpaired t-test. Pie charts depicting the different small RNA populations sequenced in the three stem cell states. Perbase sequence coverage of 30-35 nt reads across parental tRNA sequence. Anticodon positions are demarcated by discontinuous lines (Blue = LIF-treated, Grey = Wnt3+LIF- treated, Red = RA- treated mESCs). Perbase coverage of 30-35 nt reads across the 468 individual parental tRNAs plotted as heatmaps. Positional heat map of reads mapped to tRNAGlnCTG, tRNAGluTTC , tRNAValAAC, tRNAGlyGCC residues. The yellow box indicates the anticodon region.
Figure 7A. Body weight gain of male C57BL/6 mice that, after 7 weeks in HFD, started receiving treatment with CRT0066101 or water. HFD continued along with treatment. n=8. B. Organ weight (expressed as percentage of total body weight) of different fat depots and liver of male mice in panel A. C. Quantification of fat, free fluid and lean mass by nuclear magnetic resonance (NMR) of male mice in panel A. D. Glucose tolerance test 8 weeks after starting the treatment of mice in panel A. E. Insulin tolerance test 10 weeks after starting the treatment of mice in panel A. F. Western blot analysis of PKD phosphorylation in proximal jejunum of morbidly obese female patients. Samples are loaded according to fasting triglyceride levels (lower to higher). n=7. G. Scatter plot of PKD phosphorylation in jejunum (normalized to GAPDH) vs. triglycerides of morbidly obese female patients. n=7. H. Scatter plot of PKD phosphorylation in jejunum (normalized to GAPDH) vs. percentage of glycated hemoglobin (HbA1c) of morbidly obese female patients. n=7. I. Scatter plot of PKD phosphorylation in jejunum (normalized to GAPDH) vs. high density lipoprotein (HDL) of morbidly obese female patients. n=7.
Figure 6Flow cytometry analysis of SB (A), 10C (B), DLD1 (C) and HCT116 (D) cells incubated with CFSE-labeled F. nucleatum 12230 (Fn) or its fadA-deletion mutant US1 (US1) at MOI of 10-20:1 for the indicated time and immuno-stained with anti-Annexin A1 antibodies. Shown on the top panels are the density plots. x-axis, CFSE-labeled F. nucleatum or US1 (CFSE-Fn); y-axis, Annexin A1 (ANXA1). Shown on the bottom panels are the percentages of Annexin A1-positive (solid bars) or negative (clear bars) cells bound by F. nucleatum or US1 out of the total number of cells analyzed. Data are mean values ± SD. The experiments were performed in triplicates and repeated 2-3 times. *p<0.05, **p<0.01, ***p<0.001 (two-way ANOVA).
Figure 2A. Commonly and exclusively quantified proteins in bulk liver tissue, hepatic artery and portal vein. B. Cumulative protein abundance of liver biopsy, haptic artery and portal vein as a function of protein rank, with the total of the top ten abundant proteins and number of proteins that comprise four quartiles indicated. C. PCA of liver, hepatic artery, portal vein and cell lines. D. Loadings of the PCA in Panel (C) with proteins that contribute most to the variance for the three clusters annotated.
Figure 4Confocal images of ddaC neurons expressing UAS-mCD8::GFP, UAS-Nod-lacZ or UAS-Kin-lacZ and immunostained for β-galactosidase at wL3 stages. (A-C) Wild-type ddaC neurons (A) displayed normal distribution of Nod-lacZ signals in dendrites. In contrast, in msps810 mutants (B) and tacc59/tacc74 mutants (C), ddaC neurons exhibited altered Nod-lacZ distribution patterns, with highly enriched stainings in the somas and decreased signals in the dendrites. Confocal images of ddaC neurons (D-F) Kin-lacZ signals were present in axons but not detectable in dendrites of the control ddaC neurons (D), whereas part of the Kin-lacZ were mislocalized to dendrites in msps RNAi (E) and tacc RNAi (F) ddaC neurons. Asterisks indicate the location of ddaC somas. White arrows indicate the location of axons. Curly brackets mark the dendritic regions where fluorescence intensity of Nod-lacZ was measured. White arrowheads point to dendritically localized Kin-lacZ signals. G Percentages of ddaC neurons showing defective Nod-lacZ distribution in mutants or RNAi-expressing neurons. H Quantitative analysis of normalized Nod-lacZ intensity for 20 μm of major dendrite located 40 μm away from the soma. I Percentages of ddaC neurons showing defective Kin-lacZ distribution.
Figure 5IF images for total (MLC-2) and phosphorylated (pMLC-2) in PSCs, scale bar is 20 μm Quantification of staining in A. Each data point represents a cell Western blot for total and phosphorylated mlc-2 Quantification of total and active RhoA, expressed as percentage of the total RhoA in the control condition IHC images for phosphorylated mlc-2 in mice pancreatic tissues (n=5 control, n=5 2mg, and n=3 5mg). Scale bar: 100 μm Quantification of staining in E Illustration of the effect of tamoxifen on GPER activation at the cellular level (left panel) and tissue level (right panel). In PSCs, tamoxifen suppresses the activation of YAP and MLC-2 (by phosphorylation to pMLC-2) via the axis GPER/RhoA. This inhibits mechanosensing and the ability to apply endogenous forces in PSCs, which are required to maintain the myofibroblastic phenotype in PSCs. Consequently, PSCs adopt a mechanically inactive state (not myofibroblast like cell). Pancreatic tissues from KPC mice treated with tamoxifen have reduced tissue stiffness and desmoplastic reaction (decreased collagen deposition). The expression of the myofibroblast marker a-SMA is reduced consistent with the mechanical inactivation of PSCs. The recruitment of macrophages, their M2 polarization, and the invasion of cancer cells, are also reduced in these tissues
Figure 3 Figure 3. Immunodominance of PbA-derived MHC II peptides during blood stage malaria induced after spz infection (A) Experimental protocol used to analyze the ex vivo T cell responses in the spleen at day 8 and 14 following spz inoculation, with or without chloroquine (CQ) treatment as indicated. (B) IFN/TNF-double producing cells among activated CD4 T cells, after subtracting the basal level with MutuDC alone (mean +/- SEM). Asterisks show statistical significance assessed by paired non-parametric Wilcoxon tests in comparison with OVA peptide. AMA1, P = 0.027 ; TIM14, P = 0.84 ; ENO, P = 0.84 ; GAPDH.1, P = 0.002 ; MAHRP, P = 0.71; EF1α, P = 0.001 ; M1, P = 0.032 ; MSP1, P = 0.23 ; LDH, P = 0.016 ; ETRAMP, P = 0.064 ; ATPSYN, P = 0.77 ; END70, P = 0.097 ; PDI, P = 0.0052 ; GAPDH.2, P = 0.19 ; pRBC, P = 0.001. N = 15 mice pooled from 3 replicates. (C) Blood parasitemia (mean +/- SEM) monitored by flow cytometry after inoculation of 5x104 PbA.GFP spz in B6 mice, treated (open squares) or not (black squares) with CQ at day 6 and 7 pi, in order to avoid ECM without blunting infection. Day 6, P = 0.29 ; Day 7, P = 0.0013 by multiple unpaired t-tests. (D) IFN/TNF-double producing cells among activated CD4 T cells, at day 14 pi following CQ treatment (mean +/- SEM). Basal level with MutuDC alone was subtracted. GAPDH.1, P = 0.99 ; EF1α, P = 0.062 ; ETRAMP, P = 0.031 ; pRBC, P = 0.031 by paired non-parametric Wilcoxon tests in comparison with OVA peptide.
Fibroblastic FAK activity controls tumour cell migration and invasion.Figure 5(A) Representative images of three independent scratch wound assays of co-cultivated red-labelled pancreatic tumour cells with either FAK-WT (wild-type) or FAK-KD (Kinase-dead) green-labelled fibroblasts at time zero and 46 hours (corresponding to the wound closure of co-cultured FAK-WT fibroblasts with tumour cells). Cell migration (has been analyzed in detail by tracking the location of cells (n >16 cells per condition) over time for fibroblasts or tumour cells (B), measuring fibroblast velocity (C), directionality (a cell with a directionality equal to 1 is moving straightforward) (D) and migration distance (E) or tumour cell velocity (F), directionality (G) and distance (H). Values are means ± SEM of 16 tracked cells from two independent experiments. (I) 3D reconstitution of cell invasion into collagen matrix from spheroid composed of green-labelled FAK-WT or FAK-KD fibroblasts plus red-labelled pancreatic tumour cells (ratio 1/1). Shown are representative 3D reconstitution of spheroid invasion into 2mg/ml collagen I matrix obtained using Zen software. (J-K) Quantification of fibroblast (J) or tumour cell (K) invasion within collagen matrix. Values are means ± SEM of 60 tracked cells from three independent experiments.