EMBO/sd-character-level-ner huggingface dataset:
Figure 1A Overall structure (side view). EC1 and secondary structure elements in EC2 are shown in different colors, and the four TMs in blue. B Electrostatic surface representation of the structure (back view from A). The bundle region of TMs forms a tunnel that binds monoolein, and their splitting generates a lateral opening inside the membrane. C Van der Waals surface (top view) showing interactions between the TM1/2 and TM3/4 pairs near their splitting site and residues forming the lipid-binding tunnel. D Structure of the CD53 EC2 domain showing the mushroom fold with a head and stalk. The stalk helices connect to TM3 and TM4 through two hinges. E EC1 loop binds in the EC2 groove (electrostatic surface) formed by helices A and B. F Interactions stabilizing the EC1-EC2 binding.
Figure 2Expression of CA9 in formalin fixed paraffin embedded hypoxic SW620 cells by IHC. Reduced CA9 expression with siRNA confirms antibody specificity (scale bars: 200um). Expression of ; GPRC5A in formalin fixed paraffin embedded hypoxic SW620 cells by IHC. Reduced ; GPRC5A expression with siRNA confirms antibody specificity (scale bars: 200um). IHC analysis of serial sections from human colorectal tissue from patients with mesenteric ischaemia (strangulated colon). GPRC5A is co-expressed with CA9 in the colonic epithelial cells (scale bars: 50um). Quantitative RT-PCR analysis of mouse intestinal tissue. Gene expression was normalised to housekeeping gene Tbp. Raw data from three independent experiments (n=3 mice) are shown (error bars ± SEM). Tg[fli1:eGFP; vhl-/-] & Tg[fli1:eGFP] zebrafish embryos (5 days post-fertilisation) demonstrate excessive angiogenesis & increased expression of HIF target genes (scale bars: 100um). gprc5ba was induced in vhl mutant zebrafish embryos & fli1:eGFP zebrafish embryos exposed to 5% O2 (vs normoxia) for 24 hours (RT-PCR). Bioinformatic analysis of transcriptomics dataset GSE24551. Gene set analyses reveals GPRC5A mRNA strongly correlated with HIF/hypoxia gene signatures. GSEA datasets used were Semenza_HIF1_Targets (M12299) Broad_Hallmark_Hypoxia (M5891). Analysis was performed using R2 (http://r2.amc.nl). Kaplan-Meier curve following analysis of transcriptomics dataset GSE24551. Event-free survival is significantly reduced in patients with tumours expressing high levels of GPRC5A mRNA.
Figure 2A-B) Schematic of synchronisation protocol for G1/S (A) or mitotic (B) arrested cells and accompanying volcano plot of phosphorylation sites quantified. The phosphorylation sites showing an increase in the presence of the B56 inhibitor are shown in light grey. Phosphorylation sites in a protein containing an LxxIxE motif are coloured pink. C)-E) IceLogo representation of over - and underrepresented amino acid residues surrounding phosphorylation sites for the indicated experiments (letter coloring is standard iceologo color output). F) -G) IceLogo representation of over - and underrepresented amino acid residues surrounding phosphorylation sites for the experiments indicated in Figure EV2F and EV2G respectively (letter coloring is standard iceLogo color output). H) Distribution of phosphorylation site consensus motifs surrounding B56- or B55-dependent upregulated phosphorylation sites (top panel) in comparison to all phosphorylation sites (bottom panel) in the indicated experiment (proline-directed: pS/TP; basic: R/KxxpS/T, R/KxpS/T, R/KpS/T; acidic: D/E/NxpS/T, D/EpS/T, pS/TD/E, pS/TxD/E; x - any amino acid.
Figure 3A. Volcano plot showing proteins enriched after streptavidin pull-down of biotin-treated HEK293 cells stably expressing dox-inducible RNF43-BirA*. Striped line demarcates the empirical 0.01 False discovery rate (FDR) cut off. Significantly enriched Wnt/β-catenin pathway components are highlighted in red and RNF43 in orange. B. Western blot analysis of endogenous destruction complex components that co-precipitated with the indicated RNF43 cancer variants expressed in HEK293T cells. Oncogenic RNF43 truncations are indicated in red. Data information: IP; immunoprecipitation, IB; immunoblot Confocal microscopy analysis of the subcellular localization of Axin1-GFP upon expression of WT or oncogenic RNF43 (R519X). Scale bars represent 10 μm. RNF43 is visualised by Flag-staining. Asterisks indicate RNF43-expressing cells. Confocal microscopy analysis of the subcellular localization of endogenous CK1α upon expression of WT or oncogenic RNF43 (R519X). Scale bars represent 10 μm. RNF43 is visualised by Flag-staining. Asterisks indicate RNF43-expressing cells. E. β-catenin-mediated reporter activity in HEK293T cells expressing non-mutated RNF43 R519X (SLSS) or the ALAA, DLDD and ∆486-89 mutants. Average β-catenin-mediated reporter activities ±s.d. in n = 2 independent wells are shown.
Ub-positive endosomes containing Salmonella or beads are targeted by autophagy.Figure 1.(A) HeLa cells were infected with S. Typhimurium (Salmonella) for 1 h or transfected with Effectene-coated latex beads for 3 h and then subjected to immunocytochemistry for LC3 and transferrin receptor (TfR). Bar, 10 µm. (B) HeLa cells were infected with Salmonella for 1 h or transfected with Effectene-coated latex beads for 3 h and then subjected to immunocytochemistry for LC3 and galectin3. Bar, 5 µm. (C) HeLa cells were transfected with Effectene-coated latex beads for 3 h and subjected to immunocytochemistry for LC3 and Ub (top) or LC3 and p62 (bottom). Bar, 5 µm. The percentages of LC3- or p62-positive beads per Ub-positive (Ub+) or Ub-negative (Ub−) beads were enumerated. Statistical analysis was performed by Student's unpaired t test. *, P < 0.01. (D and E) HeLa cells stably expressing GFP-LC3 were transfected with Effectene-coated latex beads for 3 h. Bead-autophagosomes were fractionated as described in Materials and methods. The bead-autophagosome fraction was observed by confocal microscopy (D; bar, 10 µm) or lysed with RIPA buffer and subjected to Western blot analysis using the indicated antibodies (E). In a control sample, scraped cells were mixed with Effectene-coated beads and immediately homogenized. (F) The bead-autophagosome fraction was lysed and subjected to immunoprecipitation with an anti-Ub IgG antibody (FK2) or control IgG. Co-immunoprecipitated molecules were examined by Western blotting using the indicated antibodies. (G and H) NIH3T3 cells stably expressing mStrawberry (mStr)-Gal3 and GFP-LC3, mStr-Gal3, and GFP-p62, or mStr-Gal3 and GFP-Ub were transfected with Effectene-coated latex beads for 30 min and then washed. Live cells were observed at 1-min intervals by fluorescence microscopy. Bar, 3 µm. The time after galectin3 localization was measured for at least 30 cases for each combination (H). Statistical analysis was performed by Student's unpaired t test. *, P < 0.05; NS, not significant.
Figure 5A, B Cytosolic (black-tracing) and mitochondrial (colored-tracing) Ca2+ concentration measured using fluorescent reporters in the CHLA15 (n=29 cells)/CHLA20 (n=65 cells) and SKNBE1 (n=37 cells)/SKNBE2C (n=52 cells) pairs. ER Ca2+ release was induced by 100μM carbachol, an IP3R-agonist; time added indicated by arrow; tracings are mean +/- SD error bars. Coupling time (time between achieving 50% of maximal cytosolic and mitochondrial concentrations) is an index of ER-mitochondrial proximity and transfer efficiency; plotted below. Data information: data is derived from at least 3 separate cell transfections (biological replicates). statistical analyses were performed using an unpaired two-sided Student's t-test, with significance p<0.05. C MAM proximity was enforced in SKNBE2C cells using an ER-OMM linker construct, with Ca2+ transfer coupling time (n=57 cells) compared with cells expressing an OMM-only control linker (n=70 cells); tracings are mean +/- SEM error bars. Data information: data is derived from at least 3 separate cell transfections (biological replicates). statistical analyses were performed using an unpaired two-sided Student's t-test, with significance p<0.05. D Mitochondrial cytochrome c release for CHLA15 and CHLA20 cells following exposure to tBid or BimBH3 peptide, with or without Ru360 treatment. Data information: data points are mean of duplicate wells (SD<0.05 at all points) in a representative experiment from at least two biological replicates. E Silencing of the mitochondrial calcium uniporter (MCU) was achieved in two CHLA15-shMCU clones. F Cytochrome c release in response to tBid and BimBH3 assessed. Data information: data points are mean of duplicate wells (SD<0.05 at all points) in a representative experiment from at least two biological replicates.
Lysosomal localization of LAMP2A in Ctns−/− cells is rescued by inhibition of lysosomal proteases.Figure 7WT,Ctns−/− or Ctns−/− mouse fibroblasts treated for 20 h with either a combination of both leupeptin and chloroquine (CQ/Leu) or bafilomycin A (BafA) alone were fixed and immunostained with antibodies recognizing endogenous LAMP1 and LAMP2A proteins and samples were analyzed by confocal microscopy as described in Material and Methods. Lysosomal colocalization of LAMP1 and LAMP2A was evident in treated (middle lower and bottom panels) but not in untreated (middle upper panels) Ctns−/− cells. Scale bars: 5 μm. Inset scale bars: 2 μm.
Figure 2(A) The indicated strains were infected with SPP1 (10-6 PFU/ml), spread over MB plates, and plaque diameter was monitored after 20 hrs of incubation. Shown is average plaque diameter and SD for each strain (n ≥ 54). Plaque formation dynamic of SPP1 (B) was monitored by automated scanning Levin-Reisman et al., 2010() on a lawn of infected (10-6 PFU/ml) PY79 (WT) or ET19 (∆sigX) cells spread over an MB agar plate. Shown are average values and SD from kinetic of random plaques for each strain (n ≥ 7). Plaque formation dynamic of Phi29 (C) was monitored by automated scanning Levin-Reisman et al., 2010() on a lawn of infected (10-6 PFU/ml) PY79 (WT) or ET19 (∆sigX) cells spread over an MB agar plate. Shown are average values and SD from kinetic of random plaques for each strain (n ≥ 7). (D) PY79 (WT) and ET19 (∆sigX) cells were infected with SPP1 at either high (phages:bacteria 1:1) or low (1:20) MOI, and OD600nm was followed at 2 min intervals. Shown is a representative experiment out of 6 biological repeats, and the average values and SD of 8 technical repeats. (E) BDR2637 (Pveg-mCherry) (WT, purple) and ET191 (PrrnE-gfp, ∆sigX) (∆sigX, cyan) cells were mixed, infected with low concentrations (10-8 PFU/ml) of SPP1, placed on an agarose pad and plaque formation was followed by time lapse confocal microscopy. Shown are overlay images from mCherry (purple) and GFP (cyan) signals of the bacterial lawn captured at the indicated time points (hrs). The plaque is seen as a hole formed on the bacterial lawn. Scale bar 100 µm. Corresponds to Movie EV4. (F) ET27 (PsigX-gfp) cells were infected with low concentrations (10-8 PFU/ml) of SPP1, placed on an agarose pad, and plaque formation was followed by time lapse confocal microscopy. Shown are fluorescence from GFP (upper panels) and corresponding phase contrast images (lower panels), captured at the indicated time points (hrs). The plaque is seen as a hole formed on the bacterial lawn. Scale bar 100 µm.
Figure 8A. Network showing the functional relationship between 10 non-collinear morphological, growth and cell cycle features. The network is an undirected network highlighting the most informative connections detected by the ARACNE algorithm. The thickness of an edge represents the fraction of the networks containing this specific edge after bootstrapping the network 200 times, from 70% (thinnest) to 100% (thickest). B. Scatter plot of the normalized mean cell length and mean cell width of all 4,227 Keio strains and 240 WT replicates. Each dot represents a strain, and the gray level illustrates the density of neighbors in the vicinity of each point in the graph. The dotted contours represent the 0.5, 0.75 and 0.95 probability envelopes of the 240 WT replicates. The correlation between mean cell width and mean cell length is low (ρ = 0.06, 95% CI [0.03, 0.09]). C. Heatmap showing the mean growth rate value for data binned by both mean cell area and mean nucleoid area. The cell and nucleoid areas are strongly correlated (ρ = 0.84, 95% CI [0.83, 0.85]). The median value of αmax per bin is color-coded according to the color scale. The inset highlights the linearity of the relationship between mean cell area and mean nucleoid area. The orange dots and error bars represent the binned data, with the black line showing the best linear fit to the binned data. D. Scatter plot of the mean cell area versus the mean nucleoid area for cells with 1, 2, 3 or ≥4 nucleoids for each strain. The histogram in the inset illustrates the average proportions of cells with 1, 2, 3 or ≥4 nucleoids per strain. Although there were typically few cells in each strain with 3 or ≥4 nucleoids, at least one cell with ≥3 nucleoids was detected for 61% of the strains. E. Heatmap showing the mean growth rate value for data binned by both the mean nucleoid area and the relative timing of nucleoid separation. The mean nucleoid area negatively correlates with the relative timing of nucleoid separation (ρ = -0.48, 95% CI [-0.50, -0.45]). F. Heatmap showing the mean cell area value for data binned by both the relative timing of cell constriction and the relative timing of nucleoid separation. The relative timing of cell constriction is strongly correlated to the relative timing of nucleoid separation (ρ = 0.65, 95% CI [0.63, 0.66]).
Correlation between HCV RNA replication and appearance of double membrane vesicles.Figure 5Correlation between HCV RNA replication and appearance of double membrane vesicles.(A) Time course of spread of HCV infection in Huh7.5 cells infected with 100 TCID50/cell of Jc1. Infected cells were detected by immunofluorescence using an NS5A-specific antiserum (upper panels). The graph shown below represents the result of counting ∼200 cells for each time point to determine the percentage of infected cells. Scale bars represent 50 µm. (B) Time course of accumulation of intracellular HCV RNA in infected Huh7.5 cells. The graph shows the result of two independent experiments (3 replicas each). Whiskers indicate the minimum and maximum values. (C) Colocalization of dsRNA and NS5A in cells infected with Jc1 (10 TCID50/cell). Cells were fixed at time points specified in the left of each panel row and NS5A and dsRNA were detected by indirect immunofluorescence microscopy. DNA was stained with DAPI (blue). Boxed areas in the left panels indicate areas that are shown as enlargements in the corresponding right panels. The quantification of the degree of colocalization (Pearson's correlation coefficient) is given in the enlarged pictures. Scale bars represent 10 µm and 2 µm (left and right panels, respectively). (D) Time course of accumulation of DMVs and MMVs. For each time point, 10 cellular profiles were counted. The Mann-Whitney (non-parametric) test was applied to determine statistical significance. Error bars refer to the standard deviation. Note the striking correlation between the increase of intracellular HCV RNA and DMV number between 16 and 24 hpi.
Localization and activity of BirA*-AURKA during cell cycle A.Figure 1A. Experimental workflow for the BioID experiment. HEK293T cells stably expressing V5-BirA*-AURKA and V5-BirA* were treated with 50 μM biotin for 18 hours, harvested and lysed by denaturing lysis. Biotinylated proteins were captured with streptavidin affinity beads and analyzed by mass spectrometry. B. V5-BirA*-AURKA is expressed at near endogenous levels in the stable line. HEK293T cells and HEK293T::V5BirA*-AURKA lysates were immunoblotted with antibodies against V5, AURKA and vinculin (loading control). C. Biotinylation of the centrosome and microtubules by V5-BirA*-AURKA in different cell cycle stages. After 18 h biotin incubation, HEK293T::V5-BirA*-AURKA were fixed and stained for the fusion protein with V5 antibody, biotinylated proteins with the fluorescent streptavidin and the centrosome with gamma-tubulin antibody. DNA was stained with DAPI. Scale bar, 5 μm. D. Control and biotin-treated HEK293T cells stably expressing V5-BirA* and V5-BirA*-AURKA were lysed and biotinylated proteins were precipitated by streptavidin beads. The initial sample (initial) and captured biotinylated proteins (pulldown) were run on an SDS gel and immunoblotted with the fluorescent streptavidin and antibodies against V5 and vinculin (loading control).
Figure 4(A) Prioritized compounds proposed to have potential antiviral activities and their involvement in different modules in the viral-host PPI network. 25 compounds/drugs were identified for each of the four modules, resulting in a total of 64 distinct repurposable drugs or investigational compounds, some participating in multiple modules. The entries in the heat map display the ranking, color-coded from red (highest) to blue (lowest). The ranking was based on the proximity of their targets to proteins belonging to the modules. (B) Distribution of the same compounds/drugs in the four studied modules. Compounds belonging to selected intersections and to the viral entry module are listed. Those colored red have been experimentally tested. See the complete list in panel A and Appendix Table S5.
Figure 7(A) SARS-CoV-2 and (B) Semliki forest virus (SFV) particles were pretreated at the indicated pH for 10 min at 37°C. Viruses were subsequently neutralized with buffer at pH ~7.4 and allowed to infect Caco-2 (MOI ~0.4) and Vero cells (MOI of 0.3 and 0.8, respectively). Infected cells were then immunostained against the NP protein and SFV E2 protein, respectively, and analyzed by flow cytometry. Data are normalized to that of samples pretreated with buffers at pH ~7.4. n = 2. Samples were prepared as described in Fig. 6E but, after trypsin treatment, infected cells were subjected to buffers at the indicated pH for 5 min at 37°C and left to recover for an additional hour at 37°C. Images of microscope fields (50 < n < 120) were quantified, and fusion index was calculated as in Fig. 6F. n > 50 microscope fields were analyzed. Cell-cell fusion after trypsin treatment according to pH. The fusion is given as the ratio of the values obtained for trypsin-treated samples to those obtained for untreated samples. n = 3-4. SARS-CoV-2 particles (MOI of 0.3) were first subjected to trypsin treatment for 15 min at 37°C and then exposed to buffers at the indicated pH for 10 min at 37°C, and vice versa. Calu-3 and Vero cells were then infected at a MOI of 0.3 and analyzed by flow cytometry as described in Fig. 1D. n = 4-8 biological replicates.
AICD depends on mitochondrial fragmentation and cristae remodelling which are rescued by DRP1K38A and OPA1.Figure 2A. Protein samples (20 μg) from Jurkat cells treated as indicated were separated by SDS-PAGEand immunoblotted with the indicated antibodies. WB are representative of at least three independent experiments. B. Jurkat cells were transfected with pE-GFP and the indicated plasmids. 32h after AICD induction, viability was determined cytofluorimetrically as the percentage of GFP-positive, annexin-V-PE-negative cells. Data represent mean ± SE of five independent experiments. C. Jurkat cells were transfected as indicated. Representative reconstructions of confocal z-stacks of the mtYFP fluorescence 24h after AICD induction are shown. Scale bar, 5μm.D. Morphometric analysis of mitochondrial shape of cells transfected as in (C) is shown. Data represent mean ± SE of five independent experiments (n = 30 cells per condition in each experiment). E. AICD was induced in Jurkat cells over expressing pE-GFP and the indicated plasmids and sorted for GFP positive fluorescence. 24h after AICD induction cells were fixed and processed for electron microscopy. Representative electron micrographs of randomly selected mitochondria from cells treated as indicated are shown. White arrow points to tight cristae structure, black arrow points to remodelled cristae and to a swollen cristae junction. Scale bar, 0.2μm.F. Morphometric analysis of cristae width in cells transfected as in (E). G. Representative confocal microscopy images of cells transfected as indicated, fixed 32h after AICD induction and immunostained with an anti-cytochrome c antibody (red).H. Cytochrome c localization index was calculated from 30 randomly selected cells (per condition) transfected as in (G). Data represent mean ± SE of five independent experiments.
Netrin-1/DCC induces specific phosphorylation of phospholipase C gamma1 (PLCγ1) on Y783 by neuronal proto-oncogene tyrosine kinase Src.Figure 3(A) Co-immunoprecipitation data showed that PLCγ1 did not interact with the DCC receptor (n = 2, N = 3). (B) Immunoprecipitation assay showed that Src kinase interacted with PLCγ1. Rac1 is a positive interacting control for PLCγ1 (N = 3). (C) In vitro kinase assay confirmed that Src kinase directly phosphorylated Y783 of PLCγ1. (D) Quantification of the band intensities in (C) (N = 3). (E) Src kinase inhibitor reduced the specific phosphorylation of PLCγ1 on Y783 (arrow; Src I IC50 conc = 44 nM; N = 3). (F) Rac1 activity assay (N = 3). (G) Quantification of Rac1 activity levels in (F). (H) Representative immunoblot of cultured Plcg1f/f (n = 2, N = 3) and Plcg1f/f;Nes-Cre (n = 2, N = 3) mesencephalic neurons stimulated with conditioned media containing Netrin-1.
Figure 2(A) Cell lines endogenously expressing mutant R273Hp53 (A431 and HT29) were treated with control siRNA (ctl) or siRNA targeting exon 7 of p53, submitted to endoplasmic reticulum (ER) stress by exposure to thapsigargin (Th) and then analysed for apoptosis by incubation with propidium-iodide (PI) and FACS analysis. Data was normalized against si ctl condition, which was set to 100. (B and C) A549 (B) lung cancer cell lines stably expressing the indicated constructs were counted with Trypan blue for several days following stress stimuli (over-confluency (O-C) for A549). Data was normalized against 1st day values. Δ160p53 shows similar pro-proliferative capacities as mutant R273Hp53. On the other hand R273Hp53 lost pro-proliferative functions when deficient for Δ160p53 expression (M160AR273Hp53). Also shown is the WB of A549 cells used in (B) and stably expressing the indicated constructs. (B and C) H1299 (C) lung cancer cell lines stably expressing the indicated constructs were counted with Trypan blue for several days following stress stimuli (thapsigargin (Th) for H1299). Data was normalized against 1st day values. Δ160p53 shows similar pro-proliferative capacities as mutant R273Hp53. On the other hand R273Hp53 lost pro-proliferative functions when deficient for Δ160p53 expression (M160AR273Hp53). (D to G) H1299 cells transfected with the indicated constructs were submitted to ER stress (E and G) or cultured under regular conditions (D). Data was normalized against empty vector (E) or p53 (D) condition, which were set to 100. Cells were then analysed for apoptosis as in (A). Shown are representative data or averages + s.d. of three independent experiments (*P < 0.05, **P < 0.01, ***P < 0.005 and ^P > 0.05 compared to control ("Th + si ctl" or "-" ) or as indicated). (D to G) H1299 cells transfected with the indicated constructs were submitted to ER stress (E and G), DNA-damage by 36h 3 μM etoposide (Eto) treatment (F). Data was normalized against empty vector (E) or p53 (D) condition, which were set to 100. Cells were then analysed for apoptosis as in (A). Shown are representative data or averages + s.d. of three independent experiments (*P < 0.05, **P < 0.01, ***P < 0.005 and ^P > 0.05 compared to control ("Th + si ctl" or "-" ) or as indicated).