EMBO/sd-character-level-ner huggingface dataset:
Figure 5(A) Processing of L-PGAM5 to S-PGAM5 in depolarized mitochondria of wild type (WT), Slp2-/-, Yme11-/-, and Parl-/- MEFs. PGAM5 processing was monitored by immunoblotting at the indicated time points after inhibition of cytosolic protein synthesis with cycloheximide (CHX). A quantification of L-PGAM5 levels at different time points is shown. L-PGAM5 levels at t=0 was set to 100%. Two-way ANOVA analysis (n=3; *, p<0.05, **, p<0.01. ****, p<0.0001). Arrowheads denote intermediate PGAM5 cleavage products. (B) OMA1-mediated processing of PGAM5 in depolarized Parl-/-mitochondria. Processing of L-PGAM5 to S-PGAM5 in Parl-/ mitochondria was assessed as in (A) in the presence or absence of CCCP (20 µM), which were depleted of OMA1 by siRNA as indicated. The arrowhead denotes an intermediate PGAM5 cleavage product. SCR, scrambled. (C) Impaired PGAM5 processing in depolarized Oma1-/- mitochondria lacking PARL. Processing of L-PGAM5 to S-PGAM5 was examined as in (A) after 2 h in wild type (WT) and Oma1-/- mitochondria in the presence or absence of CCCP (20 µM, 2 h), which were depleted of PARL by siRNA as indicated. * unspecific cross-reaction. (D) OMA1 mediates accelerated processing of L-OPA1 in Slp2-/- MEFs. Processing of OPA1 and PGAM5 was monitored in Slp2-/-, Oma1-/-, and Slp2-/-Oma1-/- cells by immunoblotting after inhibition of cytosolic protein synthesis with cycloheximide (CHX). (E) OMA1 interacts with SLP2. OMA1-/-FITR293T cells ectopically expressing OMA1-myc were transfected with pcDNA5 (Control) or pcDNA5-SLP2-FLAG (FLAG). Isolated mitochondria were solubilized in digitonin and subjected to immunoprecipitation using FLAG-specific antibodies. Native eluates of the precipitate were analyzed by BN-PAGE and immunoblotting using SLP2-, YME1L-, PARL-, and myc-specific antibodies. In, input (8%); E, eluate (100%). Samples were analyzed by SDS-PAGE in parallel (lower panel).(F) Quantification of immunoprecipitation efficiencies in (E). (G) Model for the regulation of IM proteases by the membrane scaffold SLP2. SLP2 inhibits processing of L-OPA1 by OMA1 and of PGAM5 by PARL, while facilitating PINK1 cleavage by PARL, thus modulating mitochondrial morphology, quality control and cell survival.
Figure 8A. HeLa cells stably expressing CHMP4B-eGFP were transfected with the mCherry-Galectin3 plasmid. 24 h later, cells were infected with WT Coxiella burnetti. Live-cell imaging started 24 h post-infection and several time points are indicated during the next 24 h of observatio Arrows indicate Coxiella-containing vacuole (DRAQ5 labeling) becoming positive for CHMP4B-eGFP and mCherry-Galectin3. Data information: Scale bars: 50 μm B. Bacterial viability and replication assay upon TSG101 KD. HeLa cells were treated with either siCtrl or with two different siTSG101 for 48 h. Then cells were infected with mCherry-Coxiella burnetii for 48 h before lysis and serial dilutions were made to infect Vero cells. 72 h later, infected cells were fixed, DAPI stained and processed for quantitative image analysis. Between 30 and 40 fields representing more than 9,000 cells per condition of three independent experiments were analyzed. Error bars represent SD. Statistical significance was determined using one-way ANOVA test. **p-value≤ 0.01 and ***p-value≤ 0.001.
Gene expression analysis of membranal transport and apoptosis relevant genes in ASPC-1 cells.Figure 10  Quantification of mRNAs in ASPC-1 cells untreated or treated with OMP 80 µg/ml, 5-FU 5 µg/ml or the combination of both was performed at different time points throughout 24 hours, and the means of three replicates are shown for every time point. The stars indicate significant differences after 24 hours compared to control. The pro-apoptotic bad-mRNA was upregulated after 5-FU- and 5-FU+OMP-treatment. The antiapoptotic bcl-2 mRNA was downregulated by 5-FU and bcl-XL by 5-FU+OMP. OMP led to downregulation of bad and survivin and to upregulation of the mdr-1mRNA.
a) Schematic representation of the GFP–ActA–170 series of constructs.figf5(a) Schematic representation of the GFP-ActA-170 series of constructs. FL, N and C comprise ActA residues 30-612, 30-389 and 390-612, respectively. (b-e) The series of pEGFP-ActA-170 constructs was transfected into COS-7 cells. Cells were fixed with 4% Paraformaldehyde after 14 h and stained with an anti-Arp2 antibody (b), an anti-VASP antibody (c), an anti-ubiquitin antibody (d) or an anti-p62 antibody (e; red). Scale bars, 10 μm. Arrowheads indicate Arp2-, VASP-, ubiquitin- or p62-positive 170. Ub, ubiquitin.
Figure 4Analysis of the early endosome marker EEA1 in fibroblasts transduced with control or VPS16-expressing lentivirus by confocal microscopy. (A) Representative confocal micrographs. Scale bar, 20 μm. (B) Quantification of EEA1-stained puncta number and intensities (ca 80 cells analyzed for each of n=3 biological replicates). Analysis of transferrin uptake in fibroblasts transduced with the indicated lentiviruses. Representative confocal micrographs of fibroblasts fed with Alexa488-conjugated Transferrin for 30 min. Scale bar, 10 μm. Quantifications of the number and fluorescence intensities of intracellular Transferrin puncta (n=28-30 fields from 3 independent experiments). Colored horizontal bars indicate median values and whiskers 5 and 95 percentiles. Immunoblot for Transferrin receptor (TfR) levels in the indicated fibroblasts under basal and serum-starved conditions. Summary quantification of Transferrin receptor levels in non-starved fibroblasts transduced with the indicated lentiviruses, normalized to actin (ACTB) and expressed as % of controls (n=6 biological replicates). Analysis of intracellular Transferrin trafficking. Fibroblasts fed with fluorescently labeled Transferrin were co-stained with LysoTracker (G) or an antibody against LAMP2 (H). Representative images (left) of fibroblasts transduced with control virus, with magnified areas demonstrating co-localized puncta (arrowheads). Scale bars 10 μm or 2 μm (inserts). Quantifications (right) of the co-localization between Transferrin and LysoTracker or LAMP2, respectively, expressed as Pearson correlation coefficients. Colored horizontal bars indicate the median values and whiskers 5 and 95 percentiles (n=10 optical sections from 3 independent experiments).
LUZP1 is a microtubule (MT)-binding protein localized at tight junction (TJ)-associated circumferential rings (CRs).Figure 1Membrane overlay assay of Taxol-stabilized MTs using the apical junctional complex (AJC)-enriched fraction. The AJC-enriched fraction was further fractionated using ultra-centrifugation and ion-exchange chromatography with an SP-Sepharose and a Q-Sepharose column (see also Fig EV1C). The bands (marked by an arrowhead and red dots) correspond to LUZP1. 1: Ho, homogenate; 2: Sup, supernatant; 3: Pel, pellet; 4: SP-Pass, passing material through an SP-Sepharose column; 5: SP-E3(2), 2nd elution fraction through an SP-Sepharose column; 6: Q-Pass, passing material through a Q-Sepharose column; 7 to 9: Q-E3(1) to Q-E3(3), 1st to 3rd elution fractions through a Q-Sepharose column. MT co-sedimentation assay with purified Flag-LUZP1. Flag-LUZP1 directly bound MTs with a dissociation constant (Kd) of 0.78 ± 0.13 μM. A representative immunoblot and a plot in which all results are plotted with the Michaelis-Menten fitted curve are shown. n = 3. S, supernatant; P, pellet. Representative confocal micrographs of immunostained Eph4 epithelial cell sheets in the apical and sub-apical planes. LUZP1 was associated with TJs which are positive for ZO-1. Scale bar, 10 μm. A representative super-resolution micrograph of immunostained Eph4 epithelial cell sheets with the graph showing fluorescent intensities along the line. LUZP1 co-localized better with myosin IIB than with ZO-1. a.u., arbitrary units. Scale bar, 10 μm. Magnified images of Fig 1D. Scale bar, 1 μm. Representative confocal micrographs of immunostained Eph4 epithelial cell sheets in the apical plane. The apical MTs were associated with both LUZP1 and TJs. Scale bar, 10 μm (low magnification) and 1 μm (high magnification).   Representative immunoelectron micrographs of Eph4 cells. Immunogold particles for LUZP1 preferentially accumulated at the level of TJs that are positive for cingulin. MV, microvilli; AJ, adherens junction, DS, desmosome. Scale bars, 200 nm.   A schematic drawing of LUZP1 localization in association with TJs, apical MTs, and actomyosin-based CRs. Representative confocal micrographs of co-cultures of wild-type (WT) and ZO-1/-2 double knockout (DKO) Eph4 cells. ZO-1/-2 DKO cells were marked by asterisks (*). The LUZP1 junctional localization was apparently disrupted in TJ-deficient ZO-1/-2 DKO cells. Scale bar, 10 μm.   Representative confocal micrographs of transient E-cadherin knockout (KO) in WT Eph4 cells. E-cadherin KO cells were marked by asterisks (*). The LUZP1 junctional localization seemed not to change between AJ-deficient E-cadherin KO and WT cells. Scale bar, 10 μm.
Mitochondrial morphology and dynamics.Figure 7Selected proteins and complexes involved in controlling mitochondrial morphology and fusion/fission machinery and related processes of control (143B, solid lines, grey bars) and ρ0 cells (dashed lines, colored bars). A, mitochondrial contact site and cristae organizing system (MICOS: MIC60, MIC19, MIC25) and SAM complex (SAM: SAMM50, MTX2, MTX3) together forming the MIB complex Selected proteins and complexes involved in controlling mitochondrial morphology and fusion/fission machinery and related processes of control (143B, solid lines, grey bars) and ρ0 cells (dashed lines, colored bars). B, proteins involved in controlling mitochondrial morphology (orange) and dynamics (teal); Selected proteins and complexes involved in controlling mitochondrial morphology and fusion/fission machinery and related processes of control (143B, solid lines, grey bars) and ρ0 cells (dashed lines, colored bars). C, DNM1L/DRP1 and interacting proteins. For DNM1L, MTFR1 and ARL2BP, quantification of the entire migra­tion profiles (Overall) and the range above 1,400 kDa only (>1,400 kDa) are shown. Plots and gene names are in corresponding colors, except for ARL2 (dark-grey) not shown in the plot. Data are presented as mean ± SD (n=4). *p<0.05, **p<0.01, ***p<0.001 (Student's t-test).
Loss of THEM6 alters lipid homeostasis.Figure 2A, Heatmap illustrating the steady-state levels of significantly regulated lipids in THEM6 KO 22rv1 cells when compared to CTL (p ≤ 0.05, FC = 1.3). Values are expressed as log2(FC). Data information: Data reproducibility: : n = 3 independent biological experiments. EV: Empty Vector; DAG: diacylglycerol; TG: triglyceride; PC: phosphatidylcholine; PE: phosphatidylethanolamine; PI: phosphatidylinositol; LysoPC: lysophosphatidylcholine; LysoPE: lysophosphatidylethanolamine; Cer: Ceramide; SM: sphingomyelin; CE: Cholesteryl ester. B-C, Changes in lipid content (total amount) observed in THEM6 KO CRPC cells when compared to CTL. Data information: Data are presented as mean values +/- SD. Statistical analysis : *p-value < 0.05 using a 1-way ANOVA with a Dunnett's multiple comparisons test. Data reproducibility: n = 3 independent biological experiments. DAG: diacylglycerol; TG: triglyceride; PC: phosphatidylcholine; PE: phosphatidylethanolamine; PI: phosphatidylinositol; LysoPC: lysophosphatidylcholine; LysoPE: lysophosphatidylethanolamine; Cer: Ceramide; SM: sphingomyelin; CE: Cholesteryl ester. D, Heatmap illustrating the steady-state levels of significantly regulated lipids in THEM6 OE CWR22res cells when compared to EV (p ≤ 0.05, FC = 1.2). Values are expressed as log2(FC). Data information: Data reproducibility: n = 3 independent biological experiments. EV: Empty Vector; DAG: diacylglycerol; TG: triglyceride; PC: phosphatidylcholine; PE: phosphatidylethanolamine; PI: phosphatidylinositol; LysoPC: lysophosphatidylcholine; LysoPE: lysophosphatidylethanolamine; Cer: Ceramide; SM: sphingomyelin; CE: Cholesteryl ester. E, Changes in lipid content (total amount) observed in THEM6 OE CWR22res cells when compared to EV. Data information: Data are presented as mean values +/- SD. Statistical analysis: : *p-value < 0.05 using a two-tailed Student's t-test. Data reproducibility: : n = 3 independent biological experiments. EV: Empty Vector; DAG: diacylglycerol; TG: triglyceride; PC: phosphatidylcholine; PE: phosphatidylethanolamine; PI: phosphatidylinositol; LysoPC: lysophosphatidylcholine; LysoPE: lysophosphatidylethanolamine; Cer: Ceramide; SM: sphingomyelin; CE: Cholesteryl ester. F, Average Raman spectra of CWR22res CTL and THEM6 KO orthografts. Data information: Data reproducibility: : n = 4 mice per group. G-H, Quantification of tumour lipid (2845 cm-1-peak) and cholesterol (2880 cm-1-peak) content obtained from F. Data information: Centre line corresponds to median of data, top and bottom lines correspond to upper and lower quartiles. Statistical analysis Kruskal-Wallis test. Data reproducibility: : n = 6581 (CTL); 12047 (KO1); 6493 (KO2) peak intensities that were extracted from 4 mice per group.
Figure 2(A) Schematic of lysozyme application. Cells are either exposed directly to a high concentration of lysozyme (20 µg/ml) for 20 min (top) or exposed first to a short (30 min) lysozyme priming stress (1 µg/ml) before exposure to the higher concentration (bottom). (B) A priming (30 min) stress of 1 µg/ml lysozyme followed by the high lysozyme stress (20 µg/ml) improves survival. The solid blue lines are the biological repeats for the no priming experiment (n=2) whereas the solid red lines are the biological repeats for the priming experiment (n=4). (Total number of cells shown for No Priming, N = 2013 and Priming, N = 4937). (C) Surviving cells have higher PsigV-YFP levels after the initial priming stress (1 µg/ml) than perishing cells. The cumulative distributions were normalized by their maximum σV activity and baseline subtracted. Each dashed line is the mean of experiments from n=4 biological repeats. The shaded areas correspond to the mean ± s.d.. For more information on the number of repeats and cell numbers please see the supplementary text.
Figure 6 (A) Protein extracts from exponentially growing wild-type, smc5-3KE, smc5-K743,745E, smc5-4KE and smc5-K743,745R cells were prepared under denaturing conditions; 6xHis-Flag tagged SUMO (HF-SUMO) was pulled down from protein extracts to purify sumoylated species and analyzed by western blot. A strain with no HF-tag was used as control; arrow points to unmodified form of the protein, vertical bar to sumoylated forms  (B) Left, same as in (A), but using wild-type and two independent clones of smc5-7KE; right, same as in (A), but using wild-type, smc5-7KE and smc5-7KR mutant cells  (C) Quantification of Smc5 sumoylated species in pull downs from the indicated smc5-KE mutants, relative to wild-type controls from at least three independent experiments. Boxes, 25-75% data range; whiskers, total data range; black bar, median; grey cross, mean. ****P < 0.0001; ***P<0.001; **P < 0.01; *P<0.05. 1-way ANOVA, n = number of samples analyzed  (D) Co-immunoprecipitation analysis of the Smc5-Nse2 interactions. NSE2-6HA cells were transformed with centromeric plasmids expressing the indicated SMC5 alleles, and protein extracts from exponentially growing cells subjected to anti-HA immunoprecipitation. A strain with no HA tag was used as control  (E) Same as in (D), but using SMC6-6HA cells  (F) Quantification of Smc5-6HA signal from immunofluorescence on chromosome spreads prepared from exponentially growing cultures of the indicated genotypes. The mean value on wild-type spreads was arbitrarily set to 1; each dot represents one nucleus; red line, median. 1-way ANOVA, n=number of nuclei analyzed. ****P < 0.0001
POLG iPSCs manifested a partial phenotype presenting with energy depletion.Figure 2A: Representative confocal images of iPSC lineage specific differentiation into germ layers of endoderm derived hepatocytes with positive expression of ALBUMIN (red) and HNF4A (green) (a) (scale bar, 100 µm), mesodermal derived cardiomyocytes with positive expression of TNNT2 (red) (b) (scale bar, 100 µm) and ectodermal derived dopaminergic neurons with positive expression of TH (green) and MAP2 (red) (c) (scale bar, 10 µm). Nuclei are stained with DAPI (blue). B: Confocal images of mitochondrial morphology for iPSC lines with co-staining of MTG (upper panel) and TMRE (lower panel) (scale bars, 25 µm). Nuclei are stained with DAPI (blue). Flow cytometric analysis of iPSCs generated from Detroit 551, WS5A and CP2A fibroblasts for mitochondrial volume (MTG) (C, n=6, technical replicates per clone for control and CP2A; n=5, technical replicates per clone for WS5A) Flow cytometric analysis of iPSCs generated from Detroit 551, WS5A and CP2A fibroblasts for total MMP (TMRE) (D, n=6, technical replicates per clone for control and CP2A; n=5, technical replicates per clone for WS5A) and specific MMP (E, n=6, technical replicates per clone for control and CP2A; n=5, technical replicates per clone for WS5A) calculated by dividing median fluorescence intensity (MFI) for total TMRE expression by MTG. F: Intracellular ATP production in iPSCs generated from Detroit 551, WS5A and CP2A fibroblasts (n=3, technical replicates per clone for control and WS5A; n=4, technical replicates per clone for CP2A). G-H: Flow cytometric analysis of TOMM20 expression level (G, n=4, technical replicates per clone) and specific TFAM level (total TFAM/TOMM20) (H, n=4, technical replicates per clone). I: Relative mtDNA copy number in Detroit 551, WS5A and CP2A iPSCs by RT-qPCR analysis using ND1 and APP (n=5, technical replicates per clone for control; n=5, technical replicates per clone for WS5A and CP2A). Values are presented as Log2 of the ratio between the expression values of ND1 in relation to APP.
Schematic illustrating the non-cell-autonomous contributions of infiltrating retinal microglia to rod demise in inherited retinal degenerations.Figure 9In the rd10 mouse, rod photoreceptors bearing a mutation in the Pde6b protein experience cellular stress. A subset of rods undergoes apoptotic cell death (blue box) which is marked by the development of TUNEL, PI, and activated caspase-3 staining. Ramified microglia in the inner retina, sensing photoreceptor stress via unknown signals, infiltrate the outer nuclear layer (ONL) shortly after P18, expressing markers of activation (e.g., TSPO), pro-inflammatory cytokines (e.g., IL-1β), and phagocytic molecules (e.g., CD68 and MFG-E8). A subset of stressed but viable rods increases the presentation of the cell-surface "eat-me signal" phosphatidylserine (PS, green), inducing direct cell-cell contact with infiltrating microglia via dynamic microglial processes. These contacts likely result in photoreceptor axon and process retraction, which is then followed by the phagocytosis of rod somata and the clearance of living cells (orange box). Application of the vitronectin receptor antagonist, cRGD, inhibits rod phagocytosis, and also decreases microglial activation, thus reducing the positive-feedback recruitment and activation of other nearby microglia. Reduction in microglial phagocytosis, via direct inhibition or via general microglial depletion, therefore results in decreased microglial clearance via this non-apoptotic mechanism. Activated, infiltrating microglia can additionally influence and potentiate the apoptotic route for rod death via IL-1β secretion, possibly via direct signaling and/or the indirect activation of Müller cells.
Figure 9(A, B) Clonogenic assays were performed with HeLa and TRIM16KO cells, untreated or treated with As2O3 or MG132. (C) Tumor volumes of HeLa and TRIM16KO tumors at indicated time points. As2O3 is injected when tumor volume of all groups was >100mm3 (day 0 in the graph). Tumor volumes were measured on an interval as indicated. Mean ±SE, n=6 (each group), *p < 0.05, **p < 0.005, ***p < 0.0005 (ANOVA). (D) Graph shows the % tumor regression (Tumor volume on day 1 of As2O3 treatment / Tumor volume on day of sacrifice X 100). Mean ±SE, n=6 (each group), **p < 0.005 (Student's unpaired t-test). (E) Representative images of HeLa and TRIM16KO tumors formed in the nude mice in the absence and presence of As2O3. (F) Representative pictures of dissected tumors. (G) Immunohistochemistry analysis performed with KI-67 cell proliferation marker and hematoxylin nuclear stain. The graph represents the % of cells with KI-67 staining. >400 cells were counted for this analysis from three different sections from different animals, mean ±SD, *p < 0.05 (Student's unpaired t-test). Scale bar: 50 µm. (H) WB analysis of tumor tissue lysates from two different mice with antibodies as indicated. L.E, Low exposure; H.E, High Exposure. (I) Graphical representation of work presented in this study.
ILK controls lymphatic vascular growth in the heart after myocardial infarction.Figure 6LSM images of cross-sections through the heart of an adult Prox1-CreERT2;Ilk+/+ mouse (referred to as "Adult Control") and Prox1-CreERT2;Ilk∆/∆ mouse (referred to as "Adult ILK K.O.") showing the outer lateral region of the left ventricle (LV) Overall cardiac lymph vessel and blood vessel density in heart sections as determined by Lyve1-positive and CD31-positive area (normalised to total analysed myocardial area), respectively (n = 4 control and n = 5 ILK K.O. mice) LSM images of cross-sections through the heart of an adult control and ILK K.O. mouse four weeks after myocardial ischemia and reperfusion (MI/R), showing the outer lateral region of the LV. Scale bars: 100 µm Overall cardiac lymph vessel and blood vessel density in heart sections as determined by Lyve1-positive and CD31-positive area (normalised to total analysed myocardial area), respectively. (n = 5 control and n = 7 ILK K.O. mice), P = 0.051 (cardiac lymph vessel density in adult control versus adult ILK K.O.), *P = 0.025 (cardiac blood vessel density in adult control versus adult ILK K.O.)
Mec1-S1991 phosphorylation promotes replication on HU.Figure 3(A) Flow-cytometry analysis of DNA content. Asynchronous cells from the indicated strains were treated for 2h with 0.2M HU. After HU removal, recovery from replication stress was monitored by FACS (time in min). Red arrows highlight detected delays. (B) Scheme for the experimental procedure: exponentially growing cells were synchronized in G1 with α-factor and released in S phase in the presence of 0.2M HU. Newly replicated DNA was labelled with BrdU for 90 and 180 min. Analysis of replication fork progression at the single-molecule level by DNA combing. (C) Representative images of DNA fibers. Green: ssDNA, red: BrdU. Scale bar corresponds to 20 kb. (D) Graph depict the distribution of BrdU track length. WT 90 min (n=427) and 180 min (n=483), mec1-S1991A 90 min (n=309) and 180 min (n=477). (E) Analysis of DNA polymerase ε progression by ChIP-seq in S phase + HU 0.2M. Median Pol2 (DNA polymerase ε) signal at early origins (n=32) is plotted over a 30kb distance. The rate of progression is inferred from the peak at given times. (F) Graph depicting the distribution of inter-origin distances determined by DNA combing after 90 min in S phase + 0.2M HU.
Figure 1(A) Two-way unsupervised hierarchical cluster of Z-score distributions by Euclidian distance. Any miRNA mimic with a z-score ≤ -2 in at least one cell line was included. Seed sequences of each miRNA mimic are on the right. Bars correspond to arbitrary cluster boundaries. Seeds in red correspond to miRNAs with decreased expression in serous, clear cell, or endometrioid ovarian cancer relative to normal tissue (Iorio et al, 2007). (B) The histogram indicates the number of non-redundant hits binned according to the number of responsive cell lines. (C) Expression of miRNAs miR-146a and miR-505 correlated with overall survival in ovarian cancer patients. The validation cohort (n=150 samples) is shown. See Figure EV1 for the training cohort. (D) Intersection of miRNA sensitivities in the non-clonal short-term cultures and miRNAs with reduced expression in serous ovarian cancer (Iorio et al, 2007). p-value from hypergeometric distribution.