EMBO/sd-character-level-ner huggingface dataset:
Single particle diffusion analysis of CV-SU g reveals restrictive localization and diffusion of the oligomeric ATP synthase holo-complex compared to ATP synthase monomers and subcomplexes.Figure 3A,A' Trajectory maps of ATP synthase labeled at SU γ (A) and SU g (A') in single mitochondria. Insets: Detailed views of the diffusion of CV-SU γ (A) and CV-SU g (A'). Scale bars: 1 μm overview and 0.5 μm insets. B,B' Generation of transformation matrices for linearization of mitochondria to analyze the directionality of the movement. The number of individual trajectories is shown in brackets. C,C' Analysis of the step directions with respect to the longitudinal axis (±180° and 0°) and the orthogonal axis (from -90° and +90°) represented on a graduated disk. For the determination of directionality of CV-SU γ and CV-SU g, 5 successive steps were integrated. The number of analyzed steps is indicated in D and D'. D,D' Heat maps showing the step magnitude in dependence on the step direction. E Diffusion coefficients (-log D) for CV labeled at SU γ and SU g in comparison. Left: probability density function (PDF) and cumulative density functions (CDF) for D from all trajectories. Right: box and whiskers plot. Mean diffusion coefficients D (open squares) calculated from all individual trajectories shown in A and A', respectively. F Step size diagram (PDF and CDF) for steps of SU γ and SU g in comparison.
CLU is required for proliferation and cycling of PC-3 cells.Figure 1(A) Cell proliferation for PC3 cells transfected with siSCR or siCLU monitored in real time for 60h with SP Dynamic Xcelligence system (Voltage 2.5 K). Error bars represent mean ± SEM, n=4. Slopes compared by ANCOVA test, p<0.0001. (B) Left panel: Cell cycle profile of PC3 cells transfected with siSCR or siCLU. Right panel: Percentage quantification of variation in G1, S and G2/M phases. Error bars represent mean ± SEM, n=3, **p<0.01 by paired Student's t-test. (G2/M population p= 0.0058; G1 p=0.008.). Inset: Western blot with CLU antibody in PC3 cells after CLU silencing. Vinculin was used as loading control. (C) Left panel: RNA-Microarray analysis comparing PC3 cells transfected with siCLU versus siSCR and Kinexus phosphokinome microarray analysis comparing LNCaP-transfected with siCLU versus siSCR. Target genes expression is shown as fold change relative to siSCR, p ≤0.05 by an unpaired t-test for all the genes listed. Central panel: Venn diagram to graphically illustrate the overlap of mRNA and proteins involved in the regulation of mitosis in both transcriptome and phosphokinome analysis. The comparison of these 2 experiments shows modification of 4 mitosis regulators. Right panel: Western blot validating the expression levels of target proteins.
Figure 3A Compound-heterozygous mutations were identified in GFUS in one family. B (Top) Gene structure of GFUS and localization of variants. Introns length was reduced 1.5-fold. 5' and 3' UTR is shown in brown and the coding exons in green. (Bottom) Alignment and conservation of GFUS protein sequences from different species by Clustal Omega. C Partial protein structure of GFUS. Amino acid Gly204 is localized close to the GDP-D-mannose substrate binding domain whereas amino acid Ser213 is in neighborhood to the binding domain of the cofactor NADPH
Figure 4A-B, phase contrast images showing the morphological change in (A) Du145TXR or (B) MCF-7ADR cells treated with indicated agents for 72 hours. Scale bars, 25 or 50 μm (low magnification images). Data information: Results are representative of three independent experiments. C-D, endocytosis analysis of (C) Du145TXR or (D) MCF-7ADR cells treated with indicated agents for 8 hours, followed by staining with 250 μg/mL FITC-DEX for 4 hours. Scale bars, 10 μm. Data information: Results are representative of three independent experiments. E-F, phase contrast images of the morphological changes in (E) Du145TXR and (F) MCF-7ADR cells cultured in normal or serum free medium followed by treatment with indicated agents for 12 hours. Scale bars, 10 or 25 μm. The red arrows indicate vacuoles. Data information: Results are representative of three independent experiments. G-H, quantification of vacuoles in E-F. One-way ANOVA was used to analyze statistical differences. Mean with ± SD . Data information: Results are representative of three independent experiments. I-J, colony formation assay and quantification of (I) Du145TXR or (J) MCF-7ADR cells treated with indicated agents. One-way ANOVA was used to analyze statistical differences. Data information: Results are representative of three independent experiments.
Genome organization of SARS and SARS-like CoVs including Tor2, CoVZC45 and WHCV determined here.Figure 2.Genome organization of SARS and SARS-like CoVs including Tor2, CoVZC45 and WHCV determined here.
PDI Family Substrate-trapping Mutants form Mixed-disulfide Complexes with ATF6 HEK293T cells stably expressing ATF6α were either left untransfected (UT) or transfected with substrate-trapping mutants of PDI oxidoreductases.Figure 2HEK293T cells stably expressing ATF6α were either left untransfected (UT) or transfected with substrate-trapping mutants of PDI oxidoreductases. ATF6α was immunoisolated from cell lysates with mouse anti-ATF6α, separated by SDS-PAGE under non-reducing conditions and ATF6α detected by western blotting using rabbit anti-HA. Blots confirm mixed-disulfide complexes between ATF6α and ERp18, ERp57, and PDIR indicated with arrows (lanes 2, 4, 5). M, D and O refer to ATF6α monomer, dimer and oligomer respectively. Whole cell lysates of HEK293T cells expressing an ATF6α mutant containing cysteine to alanine mutations in the lumenal domain (ATF6 DM) were transfected with PDI substrate-trapping mutants. ATF6α was immunoisolated with mouse anti-ATF6α, separated by SDS-PAGE under non-reducing conditions, and ATF6α detected with rabbit anti-HA.
Complex I alterations in ∆4-CYB cells.Figure 5(A) Complexome profiles of the different cI structural modules. The graphs were generated but in this case, the peptide intensity values for the individual subunits belonging to each module were averaged to simplify the analysis The represented values are the mean ± SEM of the two reciprocal labeling experiments. (B) Complexome profiles of the cI assembly factor NDUFAF2 in WT (blue) and ∆4-CYB (red) mitochondria The represented values are the mean ± SEM of the two reciprocal labeling experiments. (C) Quantification of the total area under the curve in the profiles corresponding to each cI module (top graph). The area of the NDUFA9 subunit was calculated separately as its profile did not correspond to any of the other structural subunit modules. The plotted values are the mean mean ± SD (n=2). 2-way ANOVA with Sidak's multiple comparisons test ***p=0.0006; **p=0.0012 (N-module); **p=0.0016 (ND1-module); **p=0.0032 (ND2-module); **p=0.0057 (ND4-module); **p=0.0020 (ND5-module); *p=0.0140. (D) Quantification of the area of the N-module peak corresponding to the molecular mass of the fully assembled cI: 1,172 kDa in ∆4-CYB samples and the respirasome (cI+cIII2+cIV) SCs in #4.1 WT samples (2,110 kDa). The plotted values are the mean mean ± SD (n=2). Unpaired Student's t-test ****p<0.0001.
a depletion impairs NHEJ.Figure 4(A) Schematic diagram of the reporter construct for analyzing the DNA DSB repair by NHEJ. Incompatible DNA ends produced by I-SceI-induced DNA cleavage are repaired by NHEJ, which removes the intervening TK, resulting in GFP expression. Triangles indicate the positions of primers used to amplify a fragment encompassing the repaired junction after I-SceI cleavage. (B) Effect of Phf5a knockdown on I-SceI-induced NHEJ. Plot represents the GFP-positive cells obtained by FACS analysis after co-transfection of the I-SceI expression plasmid and the indicated siRNAs into a cell line harboring the NHEJ reporter construct. The values are presented as mean ± sd (n=3). Statistical significance was evaluated against corresponding siControl by two-tailed unpaired Student's t-test (*** p ≤0.001). (C) Genomic DNA was isolated from each transfectant and subjected to PCR using the primer pair indicated in A. (D) Confirmation of Phf5a KD in the samples by qRT-PCR. The values are presented as mean ± sd (n=3). Statistical significance was evaluated against corresponding siControl by two-tailed unpaired Student's t-test (*** p ≤0.001). (E) Representative NHEJ junctions obtained from siControl- and siPhf5a-treated samples.
Figure 5(A,B) ILC210 and non-IL-10 producing ILC2s were assessed in islet graft (A) and kidney (B) by intracellular IL-10 staining at day 5 post islet transplantation. Data shown are the mean ± SEM (n=5 per group) and an unpaired t-test was performed; ***P<0.001. ST2: suppression of tumorigenicity 2. (C) IL-10 reporter C57BL/6 mice were treated with PBS, IL-33 alone or IL-33 and IL-2C daily for 5 consecutive days. Proportion of lin-CD127+ST2+IL-10-GFP ILC210 from the kidneys at day 3 after treatment in IL-10 reporter C57BL/6 mice. Data shown are the mean ± SEM (n=4-6 per group) and a one-way ANOVA was performed; ***P<0.001. (D) Kidney ILC2s isolated from normal IL-10 reporter C57BL/6 mice were cultured with Medium only or Medium with IL-33 and IL-2C for six days. Proportion of ILC210 was assessed by flow cytometry. Data shown are the mean ± SEM (n=6 per group) and an unpaired t-test was performed; ***P<0.001. ST2: suppression of tumorigenicity 2. (E) The secreted cytokine IL-10 was analyzed via ELISA. Data shown are the mean ± SEM (n=6 per group) and an unpaired t-test was performed; ***P<0.001. (F) Kidney ILC2s isolated from C57BL/6 mice were cultured with Medium only or Medium with IL-33 and IL-2C for 30 minutes. The expression of phosphorylated STAT5 was examined in kidney ILC2s by flow cytometry. p-STAT5 (red lines) and isotype controls (gray-filled areas) are shown. Data represent the mean ± SEM of evaluations of MFI from each group (n=6 per group) and an unpaired t-test was performed; ***P<0.001. MFI, mean fluorescence intensity. (G) The kidney ILC2s were cultured with Medium, IL-33/IL-2C, or IL-33/IL-2C and STAT5 inhibitor (STAT5-IN) for three days. The secreted cytokine IL-10 was analyzed via ELISA. Data shown are the mean ± SEM (n=6 per group) and a one-way ANOVA was performed; ***P<0.001.
Figure 8Trypsin- or thermolysin-activated SARS-CoV-2 was allowed to infect A549* and Vero cells at MOIs of 0.2 and 0.3, respectively, in the continuous presence of bafilomycin A1. Infection was quantified by flow cytometry, and data were normalized to that from control samples not exposed to the inhibitor. n = 2-4 biological replicates. Binding of trypsin-activated SARS-CoV-2 to A549* and Vero cells (MOI ~0.2 and 0.3, respectively) was synchronized on ice for 90 min. Subsequently, the cells were rapidly shifted to 37°C to allow penetration. NH4Cl (50 mM) was added at the indicated time to neutralize endosomal pH and block the acid-dependent step of SARS-CoV-2 infectious penetration. Infected cells were analyzed by flow cytometry, and data were normalized to samples where NH4Cl had been omitted. n = 2. SARS-CoV-2 was first treated with trypsin or thermolysin and then allowed to infect A549* and Vero cells (MOIs ~0.2 and 0.3) in the continuous presence of SB412515 (cathepsin L inhibitor). Infected cells were quantified by flow cytometry as described in Fig. 1D, and data were normalized to samples where SB412515 had been omitted. n = 4 biological replicates.
Figure 1 Plasma levels of the 13 biomarkers belonging to clusters 3, 4, 6 (see Table 2) in healthy subjects (n=16) and PDAC patients (n=25), *p<0.01 (Mann-Whitney). Data are expressed as a scatter-plot, mean ± SEM. p-values (for each cluster) were calculated with the Wilcoxon Rank Sum Test and indicate a significant association between each cluster and PDAC.
Figure 3(A) HT29 cells were infected with the indicated Shigella strains and incubated for 8 h, and then cell lysates were subjected to immunoblotting. (B) 293T cells were transfected with Myc-tagged ospD expression plasmids. After 24 h, cells were harvested and subjected to immunoblotting. Arrows indicate cleaved RIPK1. C) Multiple sequence alignment of the OspD family: Shigella OspD3, EPEC EspL, and EHEC EspL2. Conserved amino acids are indicated by asterisks. Typical protease catalytic sites are colored in red. D) 293T cells were transfected with a series of plasmids expressing ospD3 point mutants. After 24 h, cells were harvested and subjected to immunoblotting. Arrows indicate cleaved RIPK1. (E HT29 cells were infected with Shigella WT, S325, ∆ospD3, ∆ospD3/D3 (∆ospD3 complemented with wild-type ospD3), or ∆ospD3/D3CS (∆ospD3 complemented with a protease activity-deficient mutant, in which the cysteine residue at position 64 was replaced by serine) strains and incubated for 8 h. Cell lysates and aliquots of cellular supernatants were subjected to immunoblotting F) HT29 cells were infected with Shigella WT, S325, ∆ospD3, ∆ospD3/D3 (∆ospD3 complemented with wild-type ospD3), or ∆ospD3/D3CS (∆ospD3 complemented with a protease activity-deficient mutant, in which the cysteine residue at position 64 was replaced by serine) strains and incubated for 8 h. Cell lysates and aliquots of cellular supernatants were subjected to cytotoxicity assays
Gene Set Enrichment Analysis (GSEA) showing hypoxic regulation of HIF-bound genes in (A) HKC-8, (B) RCC4 and (C) HepG2 cells.Figure 2For each cell line, genes were ranked according to hypoxic induction (0.5% hypoxia for 16 hours) in RNA-seq analyses (n = 3). Enrichment of genes (TSS) closest to each canonical HIF-α/β binding site (i.e. identified in both HIF-1α (or HIF-2α) and both HIF-1β ChIP-seq experiments) was then examined using Gene Set Enrichment Analysis (ES = enrichment score, NES = normalized enrichment score)
Fig.Figure 5(A) To ensure proper synapse formation, primary neurons were kept in culture for ten days prior to the treatment (Wan et al, 2012). Then, cells were treated with NMDA (50 µM), NMDA (50 µM) and GI254023x (5 µM), GI254023x (5 µM) alone or vehicle for 30 minutes. Total cellular mADAM10 levels remained unchanged by the treatment, which is in line with NMDA altering mainly the intracellular localization of ADAM10 by driving the protein to the synaptic membranes (Marcello et al, 2007). Densitometric quantifications of the Western Blots are shown. One-way ANOVA with post hoc Dunnett's test (*** p < 0.001, n = 7). Given are mean +/- the standard error of them mean. The mean levels of solvent treated cells were set to 1. The membrane was reprobed with the different indicated antibodies. (B) Wt neurons were kept in culture until DIV10, then the cells were treated with NMDA (50 µM), or vehicle for 30 minutes. Cell surface proteins were labeled with biotin and enriched by streptavidin pull-down. The biotinylated proteins were detected by immunoblotting. Total lysates were analyzed to compare mNrCAMs surface/lysate levels. Densitometric quantifications of the Western Blots are shown. Two-sided students t-test (** p < 0.01, n = 6). Given are mean +/- the standard error of them mean. The mean levels of solvent treated cells were set to 1. Representative Western Blots are shown.
Figure 1(A) Schematic depicting the tf-Reporter system. Both RFP and GFP fluorescence are observed throughout autophagosome formation. Upon autolysosome formation, GFP fluorescence is quenched due to the low pH of the lysosomal lumen. Corresponding changes in red:green ratio are reflective of reporter delivery to autolysosomes. (B) Dot plot indicating fraction of tf-Reporters delivered to the lysosome by ATG7-independent mechanisms. The median red:green ratio of each population was used to calculate fractional delivery according to the following formula: (ratiosgATG7-ratiosgATG9A)/(ratioNegativeCtl1-ratiosgATG9A), with the assumption that sgATG9A yields a true autophagy-null phenotype. Mean values for each reporter are indicated by a black bar. n=3 for each reporter. (C) Wild-type (WT) K562 cells and indicated deletion isolates were treated with 100 nM Bafilomycin A1 (BafA1) or DMSO for 18h and analyzed by flow cytometry for red:green ratio of tf-NBR1. Median values for each sample are identified by a black line within each violin. The red dotted line across all samples corresponds to the red:green ratio in wild-type cells. The red solid line across all samples corresponds to the ratio observed under maximally inhibited conditions (ATG9AKO cells). n = 10,000 cells. (D) Representative confocal micrographs (as maximum intensity projections) of wild-type K562 cells and indicated deletion isolates expressing tf-NBR1. Selected regions (white box) of micrographs are shown as single and merged channels from fluorescence microscopy. Red fluorescent protein (RFP), magenta; Green fluorescent protein (GFP), green; Hoechst, blue. Scale bars: large panels, 5 µm; small panels, 1 µm. All images scaled equally. (E) Correlative light and electron microscopy (CLEM) of K562 ATG7KO cells expressing tf-NBR1 under basal conditions. Analysis workflow is indicated by green arrows. White arrows indicate representative structures of interest. White boxes demarcate zoomed areas in subsequent images. NBR1, green; Hoechst, blue. Scale bar (small images), 2.5 µm. Scale bar (large images), 250 nm. (F) Presumptive model for ATG7-independent autophagy. Delivery of NBR1 to the lysosome is dependent on FIP200 (blue) and ATG9A (not shown). In the presence of LC3-lipidation, NBR1 incorporation into autophagosomes is driven by receptor interactions with LC3. In the absence of lipidated LC3, NBR1 is selectively delivered to the lysosome by a largely unknown mechanism.
Fluorescence Visualization of an ER Marker after UPR Induction.Figure 4Fluorescence Visualization of an ER Marker after UPR Induction(A) Cells treated with the UPR-inducing drug DTT (+DTT) or with no drug were visualized using a fusion protein between the translocon component Sec61 and the red-fluorescent protein "cherry." Top panels show untreated cells, and bottom panels show representative UPR-induced cells.(B) Representative images showing UPR-induced cells that contain ERAs (indicated by arrows).
Autophagic clearance of α-synuclein inclusions.Figure 2(A) Autophagic clearance of α-synuclein inclusions was verified by immunocytochemical analysis with anti-phosphorylated α-synuclein (P-αSyn) and anti-LC3 (LC3) antibodies. Confocal images reconstructed in the z-axis along the white lines are shown in the right panel (Z-axis). Blue, DAPI. Scale bar, 10 µm. Phosphorylated α-synuclein-positive inclusions were sequestrated into LC3-positive autophagosomes. (B) Similarly, this sequestration was confirmed in HEK293 cells stably expressing DsRed-LC3. The upper and lower panels show α-synuclein fibrils-and mock-introduced cells, respectively. Blue, DAPI. Scale bar, 10 µm.
Axonal transport of KIF1A cargoes by the WT and the β7 mutant E239K.Figure 2(A-F) Immunocytochemistry of DRG neurons and the corresponding quantifications of the indicated genotypes for surface TrkA (A), surface TRPV1 (B), Rab3A (C), synaptotagmin (D), DENN/MADD (E) and α-tubulin (F). TrkA and TRPV1 were visualized without permeabilization. ns, p > 0.05; *, p < 0.05; **, p < 0.01; ***, p < 0.001; Welch's t test from at least three independent cultures. Error bars show SEM. Scale bars, 2 µm. (G-J) Anterograde (red) and Retrograde (blue) velocities of ECFP-TrkA (G), TagRFP-Rab3QL (H), TagRFP-synaptotagmin (I), and mitochondria labelled with MitoBright (J). ns, p > 0.05; **, p < 0.01; ****, p < 0.0001. ECFP-TrkA, n = 25­-72; TagRFP-Rab3QL, n = 31-71; TagRFP-synaptotagmin, n = 33-62; Mitochondria, n = 21-49 moving particles were quantified from at least three independent cultures and statistical analysis performed using unpaired t test. Error bars show SEM.
a) A549 cells were infected with adenovirus harbouring shRNA targeting Atg14L, Rubicon, UVRAG or a control (luciferase).figf2(a) A549 cells were infected with adenovirus harbouring shRNA targeting Atg14L, Rubicon, UVRAG or a control (luciferase). Cell lysates were subjected to immunoblotting with the antibodies indicated. The asterisk in the Rubicon blot represents a non-specific cross-reacting band. (b) Quantification of GFP-positive dots per cell. Images were taken from cells shown in c and the number of GFP-positive dots was determined. At least 140 cells were counted per experiment and the values are mean ± s.d. of three independent experiments. (c) A monoclonal A549 cell line stably expressing GFP-LC3 was infected with adenovirus harbouring shRNA targeting Atg14L, Rubicon or control (luciferase). The cells were cultured under nutrient-rich or starvation conditions. GFP was observed by confocal laser microscopy. Scale bar, 10 μm. The full scan of the blot in a is available in Supplementary Information, Fig. S6.
Figure 7A. Huh-7 cells were depleted with control siRNA specific to firefly luciferase sequence (top panels) or with siRNA specific to γ-COP (bottom panels). Endogenous Rab18 (red) and LD (green) were stained and visualized. Bar = 10 μm. B. Huh-7 cells were treated with or without BFA for 6 hours before staining for Rab18 (red) and LD (green). Bar = 10 μm. C. TRAPPC9 were detected on Golgi but not LD surface. Huh-7 cells were permeabilized with digitonin before fixation and staining. LDs were labeled with bodipy 493/503 and pseudocolored in white. TRAPPC9 (red) and LD surface marker ADRP (green) were detected by their respective antibodies. Bar = 10 μm.
Structural flexibility analysis of the MhsT-substrate complexes.Figure 4(A,B) Structural flexibility analysis of MhsT in complex with A) L-4-F-Phe and B) L-Val as representatives of the aromatic and aliphatic substrates, respectively. Ligands are shown as magenta sticks. The analysis was visualised by atomic putty and thermal gradient representation of B-factor in Pymol. Red color indicates a high degree of disorder, while blue color indicates a low degree of disorder. In the inset, the substrates as well as the GMG loop are highlighted.