EMBO/sd-character-level-ner huggingface dataset:
a, Quantification of ATG16L1 mean fluorescence intensity by image-based flow cytometry of 10,000 cells, n = 4 (wild-type, WT), 5 (T300A).f4a, Quantification of ATG16L1 mean fluorescence intensity by image-based flow cytometry of 10,000 cells, n = 4 (wild-type, WT), 5 (T300A). Data represent 3 independent experiments. b, Quantification of caspase 3 activity upon glucose starvation of 2 × 104 cells, n = 5. Data represent 2 independent experiments. c, Quantification of mean LC3-II area of samples in a. Data represent 3 independent experiments. d, Visualization of early autophagosomes by transmission electron micrographs (TEM). Red arrows, early autophagosomes, characterized by vesicular structures with a double-membrane. Blue arrows, multivesicular lysosomal structures, identified by degraded intravesicular cargo. Micrographs representative of 10-12 images per donor, n = 4 (WT), n = 3 (T300A). A total of 29 (WT) and 26 (T300A) images containing autophagosomes was analysed. Histogram quantifies autophagic vesicles as a ratio of autophagosome:cytosolic area.
Membrane alterations induced by expression of individual HCV proteins.Figure 8Membrane alterations induced by expression of individual HCV proteins.Huh7.5 cells transfected with replicon RNAs (A, B) or Huh7-Lunet-T7 cells transfected with pTM-based expression constructs (C-H) specified in the left of each panel, respectively, were high pressure frozen 24 h after transfection, freeze substituted, embedded into epon resin and sections were analyzed by transmission EM. Representative images showing HCV-induced membrane alterations are shown. (I) Average diameter of specific vesicular structures, either DMVs or SMVs, detected in cells that had been transfected with constructs specified in the upper panels. Whiskers represent minimum and maximum values. (J) Number of vesicular structures detected in profiles of 10 transfected cells. Cells transfected with the pTM expression vector without HCV insert were used as reference. The number of vesicular structures per µm2 is given; whiskers represent minimum and maximum values. (K) Relative abundance of membranous structures. Note that only upon expression of the NS3-5A polyprotein and NS5A two different structures were observed. In all other cases, only one membranous structure was detected.
Figure 4(A) Interaction between endogenous TSC2 and Rheb in the presence or absence of lactate in COUP-TFII-silenced HCT116 cells. (B) Effect of lactate on Rheb activation in COUP-TFII-silenced HCT 116 cells. (C) Immunofluorescence analysis of staining for TSC2 and LAMP2 in HCT116 cells The degree of colocalization of TSC2 and LAMP2 is graphed as the mean ± SEM. ***p<0.001. (D) Molecular interaction between TSC2 and Rheb in the presence or absence of lactate in HA-Rheb- and/or COUP-TFII-overexpressing HCT116 cells treated with 25 nM trametinib for 24 h. (E) Effects of lactate and COUP-TFII overexpression on Rheb activation in HCT116 cells treated with 25 nM trametinib for 24 h. (F) Immunofluorescence analysis of staining for TSC2 and LAMP2 in HCT116 cells The degree of colocalization of TSC2 and LAMP2 is graphed as the mean ± SEM. ***p<0.001.
Mitochondrial sub-localization of APOPT1 A Western blot of SDS-PAGE of different fractions from 143B cells expressing APOPT1HA.Figure 4 A Western blot of SDS-PAGE of different fractions from 143B cells expressing APOPT1HA. Tot: total lysate. Cyto: post-mitochondrial fraction (cytoplasm). Mt: isolated mitochondria. Mt sol: Soluble mitochondrial fraction. Mt memb: mitochondrial membranes. CO32- pellet: Pellet after carbonate extraction with 0.1 M Na2CO3, pH 10.5 for 30 minutes. CO32- sol: soluble fraction after the carbonate extraction  B Western blot of SDS-PAGE of mitochondria used for protease protection assay after digitonin treatment. The experiment was carried out in isolated mitochondria from 143B cells expressing APOPT1HA exposed to increasing amounts of digitonin (expressed in µg) and 50 µg/ml trypsin. Complete solubilization with 1% Triton X-100 was used as a control of protease sensitivity. TOM20: Translocase of the outer membrane (OM) 20 kDa. ACO2: Aconitase 2 (mitochondrial isoform). AIF: Apoptosis inducing factor. AK2: Adenylate kinase 2. OM: outer mitochondrial membrane. IM: inner mitochondrial membrane. IMS: intermembrane space  C Western blot of SDS-PAGE of mitochondria used for protease protection assay after hypotonic shock. The experiment was carried out in isolated mitochondria from 143B cells expressing APOPT1HA incubated either in isotonic (Iso) or hypotonic buffers for 5 minutes (Hypo 5') or 15 minutes (Hypo 15') and 50 µg/ml trypsin. Complete solubilization with 1% Triton X-100 was used as a control of protease sensitivity  D N-SIM super-resolution micrographs showing 0.8 μm Maximum Intensity Projection (0.15 μm for each Z-stack) of 143B cells expressing APOPT1GFP shown in green, specific markers for each mitochondrial compartment: TOMM20 (OM), COX8A (IM), MitoTracker (IM+M) and matrix-target (mScarlet) shown in red. Scale bar: 5 μm. The graph shows Pearson's Coefficient in colocalized volume of different sub-compartment combinations with APOPT1GFP. Data show mean ± SEM (n = 5). One-way ANOVA (*P = 0.0298 and **P = 0.0076) was employed for the statistical analysis
Model for the role of Ltn1 and Ubp3-Bre5 in starvation-induced autophagy of the 60S ribosome.Figure 5.Model for the role of Ltn1 and Ubp3-Bre5 in starvation-induced autophagy of the 60S ribosome. R is a yet-unidentified specific autophagic receptor for ribosome. Ub, ubiquitin; WT, wild type.
Effects of oral administration of 2,4-dihydroxybenzoic acid (2,4-diHB) in Coq9+/+, Coq9Q95X and Coq9R239X mice and COQ9R244X patient fibroblasts.Figure 10A, B KidneyCoQ9 levels in Coq9+/+, Coq9Q95X and Coq9R239Xmice treated with 2,4-diHB (+2,4-diHB) compared with the non-treated littermate (vehicle). Statistical analysis was performed on +2,4-diHBCoq9+/+, Coq9Q95X and Coq9R239Xmice versus vehicle Coq9+/+, Coq9Q95X and Coq9R239Xmice, respectively (n = 3 for each group). C, D CoQ10 levels in COQ9R244X skinfibroblasts treated with 2,4-diHB (+2,4-diHB, blue bars) compared with the non-treated controls (vehicle, yellow bars). Statistical analysis was performed on +2,4-diHBCOQ9R244X versus vehicle COQ9R244X (n = 4 for each group).
Figure 1Growth phenotypes of the wild-type (WT, Col-0), bri1-null mutant, and two independent ubp12i/ubp13 double mutant lines grown on 1/2MS medium supplemented with 10 μM DEX (DEX medium) for 10 days and transferred to DEX medium containing 1 μM BL or mock solution (0.1% [v/v] EtOH) for additionally 8 days. Scale bars: 10 mm. Phenotype of the WT and ubp12i/ubp13 double mutant grown on DEX medium in the presence of increasing concentrations of BL in the light for 6 days. Scale bars: 10 mm.   Hypocotyl length relative to the mock control of seedlings (B). Experiments were done in triplicate (n >15 seedlings for each line).   Western blot (WB) analysis of BES1 dephosphorylation after BL treatment with the α-BES1 antibody. Total proteins were isolated from 6-day-old seedlings grown on DEX medium in the light, either in the presence of 100 nM BL or mock. PEPC was used as a loading control.   Percentage of dephosphorylated BES1 relative to the total BES1 from (D) (n = 6 biological replicates).
a,b) BJAB lymphoma cells stably expressing mCherry–GFP–LC3 were serially cultured at log phase followed by fluorescence-activated cell sorting for cells with high and low autophagic flux using the ratio of mCherry/GFP (a).figf1(a,b) BJAB lymphoma cells stably expressing mCherry-GFP-LC3 were serially cultured at log phase followed by fluorescence-activated cell sorting for cells with high and low autophagic flux using the ratio of mCherry/GFP (a). The high and low 20% were sorted (a), (a,b) BJAB lymphoma cells stably expressing mCherry-GFP-LC3 were serially cultured at log phase followed by fluorescence-activated cell sorting for cells with high and low autophagic flux using the ratio of mCherry/GFP (a). The high and low 20% were sorted (a), re-plated and treated with lysosomal protease inhibitors pepstatin and E-64d for 1 h; lysates were then immunoblotted for the indicated proteins (b). (c) Densitometry of LC3-II and p62 western blots (normalized to actin and hour 0, mean ± s.e.m., n = 3 blots from 2 independent experiments, *P = 0.051, **P = 0.0091). (d); autophagic LC3 puncta were assessed by quantitative microscopy (d); autophagic LC3 puncta were assessed by quantitative microscopy (e; punctate area per cell, mean ± s.e.m., n = 50 fields, *P = 0.010, **P = 0.053). f) Electron micrographs of HeLa mCherry-GFP-LC3 cells sorted for autophagic flux as in a. Yellow arrows denote autophagosomes; red arrows indicate autolysosomes. (g) Quantification of autophagosomes and autolysomes from electron micrographs (mean ± s.e.m., n = 50 fields). Uncropped images of blots are shown in Supplementary Fig. 6.
Fig.Figure 3(A) The chemical structures of predicted antagonists for GPR1 that showed a relative increase in trehalose in panel b are shown. (B) The distribution of the median maximum common substructure Tanimoto similarity for 10 000 random selections of 5 compounds from the Prestwick library are shown in black. The dotted red line indicates the top 5% of values, and the dotted blue line indicates the median similarity score for the predicted antagonists for GPR1 that showed a relative increase in trehalose in (Fig 3B), which are pictured in (Fig 3C). (C) The average quantified filamentous growth is shown for BY4741 and Σ1278b yeast spotted onto media supplemented with either a vehicle control (DMSO 0.1% (v/v) or the indicated compounds at a concentration of 20 µM across four biological replicates. Error bars represent the standard deviation of the mean between the averages of the replicates, and asterisks indicate p-value < 0.05 for a two-sided student's T-test comparing normalized values to the DMSO control (n = 4 biological replicates). (D) The average quantified filamentous growth is shown for BY4741 and Σ1278b yeast spotted onto media supplemented with either a vehicle control (DMSO 0.1% (v/v) or the indicated concentrations of ibuprofen. Error bars represent the standard deviation of the mean between the averages of the replicates, and asterisks indicate p-value < 0.05 for a two-sided student's T-test comparing normalized values to the DMSO control (n = 4 biological replicates). (E) The average relative amount of trehalose in ibuprofen-treated cells compared to a DMSO treatment is shown. The bar height indicates mean values, error bars indicate the standard deviation of the mean, and asterisks indicate p-value < 0.05 for a two-sided student's T-test comparing to the WT control (n = 8 biological replicates). Results are representative of 3 independent experiments.
Figure 3Top view and side cutaway view of the TssAVC cryo-EM reconstruction, shown low-pass filtered at a lower (white, transparent) and higher (royal blue, non-transparent) threshold. Possible (-60˚;60˚) range of motion of Nt2 domain relative to the CTD ring plane is shown. Top view of the ribbon diagram of TssAVC CTD model. Two interfaces are highlighted with green circles. Enlarged side view of interface between helices α9-α11 of two neighboring subunits (top right), enlarged side view of interface between α11-α12 linker of one subunit and helix α10 from the neighboring subunit based on conserved WEP-motif (bottom right). Part of Nt2-CTD linkers are highlighted with red circles. Top view (XY-plane) and side view (XZ-plane) of the TssAVC Nt2 domain cryo-EM reconstruction (pink, non transparent) shown with fitted Nt2-dimer model (left). Partial Nt2-CTD and Nt1-Nt2 linker densities are highlighted with black arrows. Top and side views of ribbon diagram of Nt2-dimer (middle). Representative 2D class averages of TssAVC particles with visible connections between Nt2-dimer and CTD (top right), representative 2D class averages of Nt2-dimer particles, Nt2-CTD linker and Nt1-Nt2 linker densities are highlighted with blue and yellow arrows (bottom right). Side and top views of putative model of TssAVC Nt2-CTD ring fitted into cryo-EM reconstruction (royal blue, non-transparent) of the distal end of TssAVC T6SS (EMD-3878). TssAVC Nt2-CTD symmetrized reconstruction shown (white, transparent), fitted Nt2-CTD model shown as a ribbon diagram.
Figure 2Flow cytometry analysis and absolute numbers of B220loCD138+ (plasmablasts) (A, B) from HET-cd19 (n=6), MaxKO-cd19 (n=8), MycKO-cd19 (n=4), and DKO-cd19 (n=5) mice. Spleen B220+GFP+ B lymphocytes were sorted and activated in vitro with LPS plus IL-4 for 72 h. Cells were stained with monoclonal antibodies against the indicated markers and analyzed by flow cytometry. *p<0.05, **p<0.01. Columns represent mean values and error bars standard deviations. Experiment representative of at least three independent experiments. Flow cytometry analysis and absolute numbers of B220+IgG1+ switched B cells (C, D) from HET-cd19 (n=6), MaxKO-cd19 (n=8), MycKO-cd19 (n=4), and DKO-cd19 (n=5) mice. Spleen B220+GFP+ B lymphocytes were sorted and activated in vitro with LPS plus IL-4 for 72 h. Cells were stained with monoclonal antibodies against the indicated markers and analyzed by flow cytometry. *p<0.05, **p<0.01. Columns represent mean values and error bars standard deviations. Experiment representative of at least three independent experiments. E Spleen B220+GFP+ B lymphocytes were stained with 7AAD and annexin-V to monitor apoptosis. 7-AAD- annexin-v- (alive), 7-AAD- annexin-v+ (early apoptotic), 7-AAD+ annexin-v+ (late apoptotic), 7-AAD+ annexin-v- (necrosis). F Relative numbers of live, early apoptotic, late apoptotic and necrotic cells from E. G CD69 surface expression in purified B220+GFP+ mature spleen cells. Sorted B cells were activated in vitro with LPS plus IL-4 for 72 h, stained and analyzed by flow cytometry. The solid grey peak represents the expression levels of CD69 marker in non-activated mature B lymphocytes at day 0. H Relative numbers of CD69+ B lymphocytes in the cultures from G (n=4). Experiment representative of at least three independent experiments. I Analysis of germinal center (GC) formation in the spleen of MaxKO-cd19, Myc-KO-cd19, DKO-cd19 and heterozygous control mice immunized with TNP-KLH. Representative images of frozen spleen sections stained with IgM (grey/blue), PNA (GC marker; red), and GFP (Max-, c-Myc- or c-Myc/Max-deficient B cells; green). Scale bar, 80µm. J Upper, Number of GCs per mouse in spleen sections of immunized MaxKO-cd19 (n=3 mice, n=186 follicles analyzed), MycKO-cd19 (n=3 mice, n=178 follicles analyzed), DKO-cd19 (n=3 mice, n=191 follicles analyzed) and heterozygous control mice (n=3 mice, n=203 follicles analyzed). Lower, Frequencies of GC (PNA+) GFP+ (deleted cells) or GFP- (non-deleted cells) in immunized MaxKO-cd19 (n=13 GCs), MycKO-cd19 (n=21 GCs), DKO-cd19 (n=27 GCs) and heterozygous control (n=62 GCs) mice. Analysis was performed at 13 days post-immunization. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.
Figure 3(A) Scheme for BrdU pulse chase experiment to track matrix cell differentiation into the upper inner lineages during anagen. The plus sign marks the starting point for TM or BrdU. (B-G) Gata6 WT and iKO skin after 3 days after TM induction and 12 hours after BrdU labeling or 5 days after TM induction and 2 days after BrdU labeling stained with markers as indicated in corresponding color.
Figure 7A. Schematic of the experiment shown in (B). B. Table summarizing whole-chromosome aneuploidy (fold over the mean) using the indicated methods to measure chromosome mis-segregation rate. Bold numbers represent significant statistical difference from the mean for each method. Orange to dark red gradient highlights chromosomes that mis-segregate (with at least one method) at a significantly higher rate compared to the mean level (weak chromosomes). Light green to dark green gradient highlights chromosomes that mis-segregate (with at least one method) at a significantly lower rate compared to the mean (stronger chromosomes). Data obtained with only one method were excluded. Im. St. = image stream. C. Plot shows the karyotype distribution of DLD-1 and RPE-1 cells in untreated or Auxin-treated condition D. Schematic of the experiment shown in (E). E. Representative image of a chromosome spreads in DLD-1 cells following mFISH and subsequently CENP-B boxes FISH. Inset shows the supernumerary (SN) chromosome. Scale bar represents 10 µm. F. Bar plot shows the mean variation in CENP-B boxes in DLD-1 cells from data from E (n = 10 cells) and whole-genome sequencing and centromere mapping ± SD. G-H. Scatter plot showing a significant positive correlation between the indicated features in DLD-1 cells (n = 1). The lines represent linear regression with 95% confidence band. Data from chr 13, 14, 21, 22, Y and SN were excluded from the analysis. I. Schematic of the experiment described in (J) and (K). J. Bars show frequency of micronuclei with or without siRNA treatment. Error bars represent the SEM of three independent experiments (n > 170 cells per replicate). K. (Left) Representative image of CENP-B box (red) staining after siRNA against CENP-A/HJURP for 48 h. CENP-B box signals at individual micronuclei are magnified in insets. Scale bar represent 5 µm. (Right) Bar graph represents the mean of the CENP-B box FISH intensity contained in the main nucleus versus the one present in the micronucleus ± SEM. Color dots represent independent experiments. (n > 43 micronuclei and 840 main nucleus) Mann-Whitney U test: ***p = 0.0001.
Figure 3(A,B) NIH3T3 cells transfected with DsRed (A) or DsRed-CA7 (B) were incubated in growth medium with 5 µM Latrunculin B for 0, 2, 5, 10, or 30 minutes, or in an equal amount of DMSO for 60 minutes. Analyses of experiments show that in cells transfected with DsRed-CA7 F-actin structures collapse more slowly (0 min: 83% "normal"; 2 min: 80%, p = 0.44; 5 min: 74%, p = 0.39; 10 min: 27%, p = 0.08; 30 min: 16%, p = 0.02; DMSO: 82%, p = 0.99; tested against 0 min with two-way ANOVA, Dunnett's multiple comparison test) than in the DsRed-transfected ones (0 min: 89% "normal"; 2 min: 48%, p = 0.14; 5 min: 31%, p = 0.04; 10 min: 2%, p = 0.001; 30 min: 0.7%, p = 0.001; DMSO: 88%, p = 0.8; tested against 0 min with two-way ANOVA, Dunnett's multiple comparison test) . For the analysis, cells were categorized to three groups as "normal", "some shape/F-actin left" and "round". The upper panel shows example images of the cells in all three categories for (A) DsRed- or (B) DsRed-CA7 transfected cells (actin visualized with Phalloidin-488).
a) Cartoon of the FPP assay illustrating the cellular localization of the Golgi marker GalT-YFP (red) and ER-bound CD3δ-CFP (green), and the position of the fluorescent protein tag relative to its respective membrane.figf4(a) Cartoon of the FPP assay illustrating the cellular localization of the Golgi marker GalT-YFP (red) and ER-bound CD3δ-CFP (green), and the position of the fluorescent protein tag relative to its respective membrane. (b) N2a cells coexpressing GalT-YFP and CD3δ-CFP were subjected to the FPP assay, as in Figure 2b c) Cartoon illustrating localization of mitochondrial EYFP-Mito (red) and Cd3δ-CFP (green). (d) N2a cells coexpressing EYFP-Mito and CD3δ-CFP were subjected to the FPP assay, as in b. Scale bars, 10 μm.
Loss of Atg7 in Myf5+ progenitors impairs brown adipose tissue (BAT) differentiation.Figure 2(A-B) mRNA for indicated genes in BAT (n=4) (c) mRNA for the indicated genes in epididymal white adipose tissue (eWAT) (n=4) (D) immunoblots for indicated proteins in BAT from 10‐month (mo)‐old chow diet (RD)‐fed control (Con) and knock out (KO) mice. Arrows depict protein isoforms. (E) immunoblots for indicated proteins in eWAT from 10‐month (mo)‐old chow diet (RD)‐fed control (Con) and knock out (KO) mice. Arrows depict protein isoforms. (F) Electron micrographs ( × 10,000 magnification) of BAT depicting mitochondria from 4‐mo‐old Con and KO mice. m, mitochondria; LD, lipid droplet; n, nucleus. (G) mRNA levels (n=4) in BAT from 10‐mo‐old Con and KO mice (H) hematoxylin and eosin (HE) and (I) Sirius Red stains in BAT from 10‐mo‐old Con and KO mice (n=3-4). Average adipocyte and LD size, and LD number in BAT are shown. Scale bar, 50 μm. (J) BAT mRNA levels from 4‐mo‐old RD‐fed Con and KO mice cold‐challenged for 75 min (n=3). Values are mean±s.e. ***P0.001.
XIAP inhibits autophagy.Figure 1(A) Representative images of GFP‐LC3 staining in HCT116 XIAP WT and XIAP KO cells with stable expression of GFP‐LC3. Quantification of LC3 punctate cells was shown on the right. The data are represented as means±s.d. of three independent experiments. (B) Endogenous LC3 expression in HCT116XIAP WT and XIAP KO cells was analysed by western blotting with anti‐LC3 antibody. The data are representative of three biological replicates. The ratio of LC3II/LC3I to actin is presented in Supplementary Figure S1A. (C) Lysates from XIAP+/+ and XIAP−/− mouse embryonic fibroblasts were analysed by western blotting with the indicated antibodies. The data are representative of two biological replicates. (D) Autophagic vesicles in HCT116 XIAP WT and XIAP KO cells were evaluated by electron microscopy. The images are representative of three biological replicates. (E) HCT116 cells were treated with Embelin as indicated. LC3‐II accumulation was determined by western blot analysis with anti‐LC3 antibody. The data are representative of three biological replicates. The ratio of LCII/LC3I to actin is presented in Supplementary Figure S1B. (F) HCT116 XIAP KO cells were treated with Embelin as indicated. LC3‐II accumulation was determined by western blot analysis with anti‐LC3 antibody. The data are representative of three biological replicates. (G) HCT116 cells were treated with Leupeptin and Embelin in the indicated combinations. Cell lysates were analysed by western blotting. The data are representative of three biological replicates. The ratio of LCII/LC3I to actin is presented in Supplementary Figure S1D. (H) MEF ATG5 WT and ATG5 KO cells were individually transfected with XIAP‐specific or control siRNAs. Forty‐eight hours after transfection, cell lysates were analysed by western blotting with the indicated antibodies. The data are representative of three biological replicates. The ratio of LCII/LC3I to actin is presented in Supplementary Figure S1E. (I) HCT116XIAP WT and XIAP KO cells were treated with 20 ‐μM Z‐VAD‐FMK or DMSO for 2 h. Cell lysates were then analysed by western blotting. The data are representative of three biological replicates. The ratio of LCII/LC3I to actin is presented in Supplementary Figure S1F. (J) HCT116 XIAP KO cells were transfected with either Flag‐XIAP (D148A/W310A) or control plasmid. Twenty‐four hours after transfection, cell lysates were subjected to western blot analysis with the indicated antibodies. The data are representative of three biological replicates. The ratio of LCII/LC3I to actin is presented in Supplementary Figure S1G.