EMBO/sd-character-level-ner huggingface dataset:
Figure 1A ROS levels were measured by flow cytometry after staining with specific oxidative stress detection dyes (CellROX green, Molecular Probes, USA) in HC (n=4) and DM (n=4) platelets. (**p=0.032 vs. HC). B Western blot analysis of ROS downstream signaling molecules p53 and phosphorylated p53 in HC (#1-3) and DM (#1-8) patient platelets. Quantification analysis on HC (n=5) and DM (n=18) individuals. (**p=0.0025 vs. HC). GAPDH served as the loading control. C Fluorescent immunostaining for phosphorylated p53 in HC and DM patient platelets. Graph indicates HC and DM platelets positive for phosphorylated p53 signal. The y-axis indicates percentage of phosphorylated p53 positive cell from total cells (**p=0.0064 vs. HC). Representative figure from n=5. D Mitochondrial membrane potential (ΔΨm) was measured by MitoTracker staining (50nM for 15 min) in HC and DM patient platelets. Graph indicates signal intensity of MitoTracker fluorescence in HC and DM platelets. The y-axis indicates fold of signal intensity in HC and DM platelet (**p=1.1608E-05 vs HC). Signal intensity of each group was converted to fold change compared with HC values. Representative figure from HC (n=6) and DM (n=4). E ΔΨm and platelet apoptosis were measured by flow cytometry analysis. ΔΨm was detected using TMRM and apoptosis was assessed with Annexin-V (PS externalization). Representative figure from n=3. F Western blot analysis of apoptosis-related pp53, p53, cytochrome c, and active caspase 3 in HC and DM platelets. Tubulin was used as the loading control. Representative figure from n=3. G Western blot analysis showing release of cytochrome c (Cyto c) into the cytosol in DM. Representative figure from n=3. H EM of platelets mitochondria from HC and DM patients demonstrating examples of mitochondria damage in DM platelets (DM) compared to a typical healthy mitochondria (HC). Arrows indicate disrupted outer membranes.I Quantification of mitochondria in HC (n=33) and DM (n=17) patient platelets. The y-axis indicates average number of mitochondria in HC and DM single platelet EM view (**p=5.50811E-05 vs. HC).J Quantification of healthy and damaged mitochondria in HC and DM platelets.
Figure 2 (A) Schematic representation of the RAPID-release technique. Addition of rapamycin results in recruitment and activation of an auto-inhibited TVMV protease, leading to the release of EGFP-labelled histones from their mitochondrial tether. TVMV - Tobacco Vein Mottling Virus, FKBP - FK506-binding protein (FKBP12), FRB - FKBP12-rapamycin binding domain, OMM - Outer Mitochondrial Membrane, OMP25 - 25 kDa outer-membrane immunogenic protein (C-terminal helix), AI - Auto-inhibitory peptide  (B) A representative cell showing release of tethered EGFP. Scale bar represents 5 μm  (C) Quantification of EGFP release. Single cells are represented as grey traces. The cell displayed in (B) is highlighted with red circles. The blue line represents a fit to an exponential decay model. (D) Rate constants from exponential decay functions of the traces shown in (C) plotted against the expression level of protease relative to EGFP substrate. The blue line represents a linear regression model with the R2 value shown  (E) Representative cells showing release of H4-EGFP and H3.1-EGFP. Scale bar represents 5 μm  (F) Nuclear import rate and cleavage rate of H3.1-EGFP and H4-EGFP. Single cells are represented as grey traces. Traces from cells displayed in (E) are highlighted with circles. (G) Import rate plotted against cleavage rate. Values represent those shown in (F), with the cells shown in E highlighted with red circles. Linear regression models are shown with blue lines. The mean R2 and slope (m) are shown for the 11 cells quantified in (F), +/- relates to standard deviation
Figure 6Negative geotaxis climbing assays in Drosophila (flies). Time required for 50% of flies to climb to the target line before or after 2.5 hr of continuous climbing exercise (A) in the yw strain with muscle-specific STIM knockdown using two independent RNAi sequences (RNAi26-1 or RNAi8-4) compared to GFP-expressing controls, or (B) in the w1118 strain with muscle-specific STIM knockdown using RNAiGD16187 compared to non-targeted RNAiGD12145 controls. Time to fatigue, when flies were no longer able to climb, during a 3-min period after negative geotaxis stimulation in STIM whole-body knockout flies overexpressing wild type (WT) or mutant (S257A or S257E) human STIM1-mRuby3.
Figure 4A. HEK293T cells with the indicated genotypes were loaded with 400 μM oleic acid for 24 h before staining with bodipy 493/503. Bar = 10 μm. B. Wildtype or TRAPPII deleted cells were loaded with oleic acid and stained with bodipy 493/503 (green) and endogenous Rab18 by immunofluorescence (red). Bar = 10 μm. C. DsRed-Rab18 was transfected into wildtype (top panels and insets) or TRAPPII deleted HEK293T cells (bottom panels and insets) that were loaded with oleic acid and stained with bodipy 493/503 (green). Bar = 10 μm. D. Homogenates of wildtype or TRAPPII deleted cells were subjected to sucrose gradient fractionation. Fractions 1 and 2 were enriched with LDs. The presence of Rab18, TRAPPC9, TRAPPC2, LD marker TIP47, and ER marker Calnexin were detected by SDS-PAGE followed by immunoblotting.
Molecular docking simulation followed by mutational studies.Figure 5A The most optimal docking model for the AdipoR1-P5 complex. P5 is shown in magenta sticks. Each amino acid of AdipoR1 in yellow was mutated in the following experiments. B The magnified view for the detailed AdipoR1-P5 complex interface (magenta, P5; yellow, amino acids of AdipoR1); ECL, extracellular loop of AdipoR1. Pulldown of Flag-fused AdipoR1 input (residues 89-375) with amino acid mutation(s), as indicated, with GST-P5. The relative pulldown efficiency is expressed as the ratio of the band intensities of bound AdipoR1 to those of AdipoR1 (residue 89-375) inputs, as indicated (n = 3 technical replicates / group). Pulldown of Flag-fused AdipoR1 input (residues 89-375) with amino acid mutation(s), as indicated, with GST-P5. The relative pulldown efficiency is expressed as the ratio of the band intensities of bound AdipoR1 to those of AdipoR1 (residue 89-375) inputs, as indicated (n = 3 technical replicates / group). F Pulldown of AdipoR1 (residues 89-375) with GST-P5 or GST fused to two mutant peptides (GLYDF and GLYAF). The relative pulldown efficiency is expressed as the ratio of the band intensities of bound AdipoR1 to those of AdipoR1 (residue 89-375) inputs, as indicated (n = 3 technical replicates / group).
Figure 8(A) Cortical and hippocampal mixed neuronal cultures were transfected with a GFP construct at 12 DIV and treated with poly(I:C) (pIC) at 17 DIV for 24 h. Spine morphology was examined at 18 DIV. Three secondary dendrites of each neuron were selected to analyze the spine density, the width of the spine head and the spine length. Mean values SEM of representatives of three independent experiments are shown. For the saline group, 15 neurons/45 dendrites were examined; for the poly(I:C) group, 16 neurons/48 dendrites were examined. Data were analyzed by unpaired t-test. Scale bar, 20 μm in right upper panel and 5 µm in lower left panel. *** P < 0.0001. (B) Schematic of the experimental procedure for in vivo spine morphology analysis in C and D. Representative images of somatosensory cortical layer 5 neurons and a high magnification image of a secondary dendrite are shown. Scale bar, 100 μm in the brain section (black) and 10μm in the dendrite segment (white). (C, D) Spine morphology of P21 Thy1-Yfp (C) and Tlr3-/-; Thy1-Yfp mice (D) after poly(I:C) stimulation at P4 and P5. One secondary dendrite of an apical dendrite of each somatosensory layer 5 cortical neuron was selected to examine the spine morphology. Three mice were analyzed for each group. Saline-treated Thy1-Yfp, 44 neurons; poly(I:C)-treated Thy1-Yfp, 43 neurons; saline-treated Tlr3-/-;Thy1-Yfp, 46 neurons; poly(I:C)-treated Tlr3-/-;Thy1-Yfp, 50 neurons. Mean values SEM are shown. Data were analyzed by unpaired t-test. Scale bar, 5 μm. *** P < 0.0001.
Figure 3IFM images of KIF13A-YFP transfected control and Rab22A-, BLOC-1Mu-, BLOC-2HPS6- knockdown HeLa cells. Cells were stained for AP-1 (γ) or internalized with Tf-Alexa fluor 594 arrowheads and arrows point to the KIF13A-/Rab22A-positive tubular REs and E/SEs respectively. Scale bars: 10 μm Graphs represent the measurement of KIF13A-positive TN (B) and TL (C) in HeLa cells of Fig 3A. n=3. nc: total number of cells. nt: total number of tubules Immunoblotting analysis of proteins in control and Rab22A-, BLOC-1-, BLOC-2-depleted HeLa cells. *, non-specific bands. Protein band intensities were quantified and indicated on the gels. IFM images of mCherry-Rab22A and KIF13A-YFP cotransfected control and BLOC-1Mu-, BLOC-2HPS6- knockdown HeLa cells ), arrowheads and arrows point to the KIF13A-/Rab22A-positive tubular REs and E/SEs respectively. Scale bars: 10 μm
a, COS7 cells expressing GFP–SEC61&bgr and STX17 were immunostained for TOMM20.f3a, COS7 cells expressing GFP-SEC61b; and STX17 were immunostained for TOMM20. b, HeLa cells expressing GFP-ATG14 were immunostained for STX17. c, Immunoblot of subcellular fractions of HEK293 cells. d, STX17 knockdown COS7 cells expressing GFP-ATG14 and RFP-SEC61b; were immunostained for TOMM20. Error bars denote s.d. e, Immuoblot of subcellular fractions of STX17 knockdown (using two STX17 siRNAs, STX17-1 and STX17-2) HEK293 cells expressing GFP-DFCP1. f, Co-immunoprecipitation of STX17 and ATG14 using HeLa cells expressing Flag-ATG14 and Myc-STX17. g, Pull-down of ATG14 complex from HeLa cells expressing Myc-STX17 or Myc. Scale bars, 5 µm (a, b) and 2 µm (d).
Figure 1(A) Isolation of FTCD from rat liver extract. The p47(1-170) fragment, which does not contain the p97-binding site, was immobilized on beads and incubated with rat liver extract. Bound proteins were fractionated by SDS-PAGE, and stained with CBB. (B) FTCD binds directly to p47. GST-tagged p47 (0.54 μg) or GST-tagged p37 (0.45 μg) was incubated with His-tagged FTCD (0.80 μg), and then isolated on glutathione beads. The blots were probed with antibodies to FTCD and GST. (C) FTCD and p47 form a complex in cells. HepG2 cells were solubilized and incubated with anti-p47 antibodies. The immunoprecipitates were fractionated by SDS-PAGE, followed by Western blotting with antibodies to p47 and FTCD. (D) FTCD, p47 and p97 form a complex in cells. HepG2 cells were solubilized and incubated with anti-FTCD antibodies. The immunoprecipitates were fractionated by SDS-PAGE, followed by Western blotting with antibodies to FTCD, VCIP135, p97, p47 and syntaxin5. (E) p47 binds to the CD domain of FTCD. GST-tagged p47 (0.50 μg) was incubated with either His-tagged FTCD(1-326) (FT, 0.88 μg) or His-tagged FTCD(327-541) (CD, 0.63 μg). The blots were probed with antibodies to His-tag and GST. (F) p47(E86K, E88K) does not bind to FTCD. GST-tagged p47wt/mutant (0.50 μg) was incubated with the indicated His-tagged proteins (p97, 1.0 μg; FTCD, 0.75 μg). The blots were probed with antibodies to p97, FTCD and GST. (G) FTCD(R382A), which lacks binding ability to polyglutamates, does not bind to p47. GST-tagged p47 (0.35 μg) was incubated with His-tagged FTCDwt/mutant (0.53 μg). The blots were probed with antibodies to His-tag and GST. (H) p47 binds to FTCD in Golgi membranes. Salt-washed Golgi membranes were first incubated with either GST-tagged p47wt, p47(E86K, E88K) or p47(F253S). The isolated membranes were solubilized, and then GST-tagged p47wt/mutants and their binding proteins were recovered with glutathione-beads. The bound proteins were assayed by Western blotting with antibodies to FTCD and GST.
Figure 5A Yeast two hybrid (Y2H) assays of the extracellular juxtamembrane domains of HERK1 and ANJ (HERK1exJM and ANJexJM, respectively) with LRE (residues 23-138; signal peptide and C-terminal domains excluded). B Y2H assays with the extracellular domains of HERK1, ANJ and FER (HERK1-ECD, ANJ-ECD and FER-ECD, respectively). Ø represents negative controls where no sequence was cloned into the activating domain (AD) or DNA-binding domain (BD) constructs. -L-W-H, growth medium depleted of leucine (-L), tryptophan (-W) and histidine (-H). C Co-immunoprecipitation of HA-LRE with HERK1-GFP or ANJ-GFP following 2 days of transient expression in N. benthamiana leaves. D Co-immunoprecipitation of FER with HERK1-GFP in Arabidopsis seedlings expressing pFER::HERK1-GFP. Numbers indicate molecular weight marker sizes in kDa. Assays were performed twice with similar results. CBB refers to Coomassie Brilliant Blue staining of total proteins.
a, Electron micrographs of cultured hepatocytes.figf3a, Electron micrographs of cultured hepatocytes. dm, double-membrane cytosolic vesicles. Arrows indicate membranes in LDs. Insets show the double membrane in nucleus (N), lipid-containing vesicles (L) and mitochondria (mit), but not in LDs. b, Mouse liver LC3 immunogold. Insets show higher magnification. Arrowheads indicate gold particles (black) and LC3-labelled bilayer membranes (white). c, Percentages of autophagic vacuoles (AVs) containing only lipid (Lipids, *), other cargo (Other) or mixed cargo (Mixed) in cells treated as in b (*P  0.01, **P  0.001 with cells in RM, n = 46). Left: representative examples. Error bars, s.e.m.
Figure 7Left panel: In a cell that expresses degradable WT-β-catenin, the active GSK3β phosphorylates β-catenin at the Ser-33 and Ser-37 residues (number 1), followed by the recruitment of β-TrCP, which leads to polyubiquitination-dependent proteasomal degradation of β-catenin (number 2 and 3). As a second step (II), the released RAS by prior β-catenin degradation is subjected to phosphorylation by GSK3β at Thr-144 and Thr-148 (number 4), recruits β-TrCP (number 5), and is subsequently degraded through proteasome (number 6). Right panel: In a cell that expresses nondegradable MT-β-catenin, RAS protein masked by MT-β-catenin at the phosphorylation sites cannot be phosphorylated by GSK3β and costabilized with β-catenin. Blue stars represent mutation sites (Ser-33 and Ser-37) of β-catenin. Expanded View Figure legend
Figure 3A. Immunostaining of Tom20 and Drp1 in N2As depleted of Ifnb (δIfnb) by CRISPR/Cas9 and matching nontargeting control (NTC). B. Immunoblot of Drp1 from cell fractions of wild-type (WT), δIfnb, and NTC N2As. Purity of cytoplasmic (C) and mitochondrial fractions (M) were evaluated by immunoblotting of tubulin and Tom20, respectively. Immunoblot of Drp1 from cell fractions of wild-type (WT), δIfnb, and NTC N2As. Purity of cytoplasmic (C) and mitochondrial fractions (M) were evaluated by immunoblotting of tubulin and Tom20, respectively. C. Quantification of B. D. Quantification of anti-pDrp1 immunohistostains in different regions of the brain. Images shown in Fig EV3. E. Immunoblot of phospho-S622 Drp1 in cortices and brainstem of 3-month-old Ifnb+/+ and Ifnb-/- mice and quantification. F. Phospho-S622-Drp1 immunoblot and quantification in Ifnb+/+ and Ifnb-/- CN cultures. Vinculin was used as a loading control. Error bars are SEM of 3 independent experiments. G. Kinetics of Drp1 phosphorylation on S622 on rIFN-β treatment in wild-type CN cultures. Error bars are SEM from 3 independent experiments. H. Kinetics of Drp1 dephosphorylation at S643 on rIFN-β treatment in wild-type CN cultures. PD-L1 was used as a positive control of rIFN-β activity. I. MFN2 and OPA1 immunoblots and quantification in Ifnb+/+ and Ifnb-/- CN cultures and quantification. Vinculin was used as a loading control. Error bars are SEM from 3 independent experiments. J. Optineurin and Tom20 immunofluorescence in Ifnb+/+ and Ifnb-/- MEFs transfected with Drp1 or phosphomimetic Drp1. K. Mean mitochondrial section area in Ifnb+/+ and Ifnb-/- MEFs transfected with Drp1 or phosphomimetic Drp1. Error bars are SEM from 10 transfectants. Optineurin and Tom20 immunofluorescence in Ifnb+/+ and Ifnb-/- MEFs transfected with Drp1 or phosphomimetic Drp1. L. Quantification of optineurin staining in J.
Figure 4Representative images of 20 tissue types and histological structures stained on consecutive sections with immunohistochemistry using two antibodies targeting human ACE2 protein (brown), and counterstained with hematoxylin (blue). Most intense antibody staining was observed in microvilli of the intestinal tract and renal proximal tubules, in membranes of gallbladder epithelium, epididymis epithelium, testicular Sertoli cells and Leydig cells, a subset of glandular cells in seminal vesicle and cytoplasm of cardiomyocytes, with HPA000288 also staining the cardiac muscle fibers, while MAB933 only showed staining in a few cells. Distinct ACE2 staining for both antibodies was also present in cornea and conjunctiva of the eye, interlobular pancreatic ducts, as well as in placental villi, both in cytotrophoblasts, syncytiotrophoblasts, and also in extravillous trophoblasts, while placenta decidua was negative. ACE2 staining could be observed at the base of ciliated fallopian tube epithelium, however only for one of the antibodies. Note that ACE2 protein expression was less prominent in the crypts of the mucosal intestinal layer. ACE2 also stained positive in endothelial cells and pericytes in several tissues, see Fallopian, thyroid, parathyroid, adrenal gland, pancreas and heart. Scale bar = 50µm. Scale bar in dashed squares = 10 µm. (Brunner = Brunner's gland, EVT = extravillous trophoblasts, endo=endothelial cells).
FXR1 regulates G1 cell cycle progression.Figure 9A HeLa cells were transfected with the import/export reporter plasmid XRGG-GFP, treated with the indicated siRNAs and synchronized in early G1 phase by Monastrol release. Dexamethasone was added for 3 hours to induce XRGG-GFP nuclear import. Following wash-out the nuclear export of XRGG-GFP was analysed by live video spinning disk confocal microscopy. The selected frames of the movies are depicted in Figure EV5C. The percentage of cytoplasmic XRGG-GFP over time was quantified in Figure EV5D and quantifications of individual cells from the 20 and 30 minutes time points are depicted in (A), 199 cells were analysed (mean ±SD, *P < 0.05; **P < 0.01; N = 3). B HeLa cells stably expressing GFP-Nup133 were treated with the indicated siRNAs, synchronized by double thymidine block, released for 12 hours and analysed by immunofluorescence microscopy. The magnified framed regions are shown in the corresponding numbered panels. C, D Asynchronously proliferating HeLa cells were treated with indicated siRNAs and analysed by immunofluorescence microscopy (C). The percentage of p-Rb positive cells was quantified in (D), 2800 cells were analysed (mean ±SD, **P < 0.01; N = 3). Asynchronously proliferating HeLa cells were treated with indicated siRNAs, incubated with EdU during 30min and analysed by immunofluorescence microscopy. The percentage of p-Rb and/or EdU positive cells was quantified in (E), 2100 cells were analysed (mean ±SD, *P < 0.05; **P < 0.01; N = 3) F Asynchronously proliferating HeLa cells were treated with indicated siRNAs, incubated with EdU during 30min and analysed by immunofluorescence microscopy. the percentage of p-Rb and/or cyclin B positive cells was quantified in ( G A hypothetical model how Fragile X-related proteins spatialy regulate nucleoporin condensation. FXR proteins (blue) interact with cytoplasmic soluble Nups (red circles) and dynein (green) and facilitate their localization to the NE during early G1. This function of FXR proteins inhibits formation of aberrant cytoplasmic Nup assemblies, the Cytoplasmic nucleoporin Granules (CNGs), contributing to the equilibrium of NE-NPCs and driving the G1-specific protein export and maintenance of nuclear shape and cell cycle progression. Silencing of FXR proteins, for instance in FXS patients, leads to the accumulation of CNGs, nuclear atypia, protein export defects, and defects in cell cycle progression, which may contribute to the pathology of FXS.
Figure 8(A) Western blots and quantification of VEGFR2 phosphorylation levels relative to total VEGFR2 levels in WT and EVL-/- endothelial cells under basal and VEGF-stimulated conditions (80 ng/ml); n = 5 independent cell batches, error bars represent SEM; *p<0.05, one-way ANOVA with Bonferroni's multi comparison test. (B) Western blots and quantification of ERK1/2 phosphorylation levels relative to total ERK levels in WT and EVL-/- endothelial cells under basal and VEGF-stimulated conditions (80 ng/ml); n = 6 independent cell batches, error bars represent SEM; *p<0.05, one-way ANOVA with Bonferroni's multi comparison test. (C) ERK1/2 phosphorylation in P5 retinas of wild-type (WT) and global EVL-/- mice. Retinas were fixed and stained with isolectin B4 (IB4, green) to visualize endothelial cells and antibodies directed against phospho-ERK1/2 (red). Representative images from three independent experiments are shown. Scale bars, 100 µm.
Figure 5A - Schematic view of the hexosamine biosynthesis pathway. Production of glucosamine-6-P by GFPT is the first and rate-limiting step of the pathway that produces UDP-GlcNAc. HK: hexokinase; G6PI: glucose-6-P isomerase; GFPT: glutamine:fructose-6-P amidotransferase; GNA: glucosamine-6-P N-acetyltransferase; PGM3: phosphoglucomutase 3; UAP: UDP-N-acetylglucosamine pyrophosphorylase; OGT: O-GlcNAc transferase OGA: O-GlcNAcase; GlcNAc: N-acetylglucosamine. B - Endocervical epithelial cells were pre-treated or not with 40 μM CP4d for 2 h before addition of the indicated concentration of ionomycin (or an equivalent volume of DMSO) and 0.5 µM BP. Six hours later whole cell lysates were analyzed by western blot. The membrane was first blotted with HRP-conjugated streptavidin to detect TG2 activity, then extensively washed and probed with anti-O-GlcNAcylation antibody followed with HRP-conjugated secondary antibodies. Last the membrane was probed with anti-actin as a loading control. C - The same experimental procedure as described in (B) was applied to HeLa cells treated for 48 h prior to ionomycin treatment (8 µM) with siRNA control or directed against TG2.
Figure 2A) Cells of the indicated genotypes were pre-cultured in medium with 10mM ethanolamine and 10mM choline and subsequently in medium lacking them for 5 hours, before being diluted in the indicated medium for growth curve analysis. Emp. vec. refers to cells bearing an empty vector. Data shown are the mean ±SEM of three independent replicates, except for Psd(MIM) (two experiments). B) Five-fold serial dilutions of strains of the indicated genotypes (wt cells and choppΔ cells harboring empty vectors or choppΔ cells expressing chimeric Psd and Pmt enzymes). Cells were grown on SD-HIS-LEU (-HL) medium (KennedyOFF) or SD-HL containing ethanolamine and choline (KennedyON). C) Thin layer chromatography (TLC) analysis of steady-state phospholipid profiles of cells of the indicated genotypes. Where Psd and/or Pmt are missing (-), choppΔ cells were grown in the presence of ethanolamine and/or choline. Bands corresponding to the phospholipids PA, PS, PE, PI and PC are indicated.