EMBO/sd-character-level-ner huggingface dataset:
Active Rap2b is decreased to allow the autophagy response induced by Hla and S.Figure 11(A) HeLa cells were incubated for 4 h with complete medium in the presence (Hla) or absence (Ctr) of 10 µg/ml α-hemolysin or they were infected for 4 h with the wt strain of S. aureus (wt) or the α-hemolysin deficient mutant (Hla−). Afterwards, cells were lysed with sample buffer and the samples were subjected to Western blot analysis using a rabbit anti-Rap2b and the corresponding HRP-labeled secondary antibody. The bands were subsequently developed with an enhanced chemiluminescence detection kit. A quantification of the bands intensities with the Adobe Photoshop program is shown in the lower panel. These data are representative of two independent experiments. B) HeLa cells were incubated for 4 h with complete medium in the presence (Hla) or absence (Ctr) of 10 µg/ml α-hemolysin or they were infected for 4 h with the wt strain of S. aureus (wt) or the α-hemolysin deficient mutant (Hla−). Cells were disrupted and whole cell lysates were subjected to pull-down assays using GST-Ral-GDS-RBD-sepharose. The levels of GTP-bound Rap2b were determined as was described in Materials and Methods by Western blot analysis using a rabbit anti-Rap2b and the corresponding HRP-labeled secondary antibody. The bands were subsequently developed with an enhanced chemiluminescence detection kit. The band intensities were quantificated with the Adobe Photoshop program is shown in the lower panel. * p<0.05 (paired Student's t-test). These data are representative of two independent experiments.
Baseline characterization of mice with cardiac-specific LXRα overexpression.Figure 1A Murine LXRα DNA construct for the generation of transgenic (LXRα-Tg) mice. B-D Left ventricular LXRα expression in Wt and LXRα-Tg mice aged 12 weeks. (B) Western blot of LXRα in liver and LV tissue. B-D Left ventricular LXRα expression in Wt and LXRα-Tg mice aged 12 weeks. (B) Western blot of LXRα in liver and LV tissue. Quantification of (C) mRNA expression (normalized to 36b4; n = 6-8/group, *P < 0.00001 versus Wt) and (D) protein expression (normalized to GAPDH; n = 4/group, *P = 0.0001 versus Wt). E LV weight to tibia length ratios (LV/tibia); n = 15/group. *P = 0.02. F Assessment of mean arterial pressure (MAP) with in situ catheterization shows no significant differences. G Representative Masson's trichrome and wheat germ agglutinin (WGA)-FITC staining in cross-sectional LV, scale bars = 100 μm and 10 μm, respectively. H Relative mRNA levels of known LXRα-associated and target genes assessed with RT-PCR; n = 8/group. *P = 0.0003, #P = 0.0004, §P = 0.043, †P = 0.0009 versus Wt.
Figure 1A Diagrammatic sketch of in-situ binding kinetic assay and functionalization of RBC. B Representative adhesion frequency (Pa) vs. contact duration (tc) curves forNKG2D expressing NK cells (n ≥ 3) in contact with RBCs (n ≥ 3) coated with a ligand (MICA in red, MICB in orange, ULBP1 in green, or ULBP3 in blue) at different contact durations, fitted by non-linear in-situ binding kinetics model Site densities of NKG2D (mr) and its ligands (ml) are indicated. Data information: Error bars are mean ± SEM for at least three independent biological experiments where *P < 0.05, **P < 0.01 (two-tailed unpaired t-test). In-situ force-free affinities (C), on-rates (D) of NKG2D binding with indicated ligands from mammalian cells. The in-situ force-free kinetics were obtained from fittings with in-situ binding kinetics modeling in (B). Data information: Every dot represents one independent binding experiment. Error bars are mean ± SEM for at least three independent biological experiments where *P < 0.05, **P < 0.01 (two-tailed unpaired t-test). In-situ force-free off-rates (E) of NKG2D binding with indicated ligands from mammalian cells. The in-situ force-free kinetics were obtained from fittings with in-situ binding kinetics modeling in (B). Data information: Every dot represents one independent binding experiment. Error bars are mean ± SEM for at least three independent biological experiments where *P < 0.05, **P < 0.01 (two-tailed unpaired t-test). F Detection range comparison in affinity measurement of NKG2D and indicated ligands between in-situ and in-solution assay. Bars in different colors are the ratios of the affinities of indicated ligands divided by that of ULBP1. Data information: In-solution affinities of proteins purified from E. coli In-solution affinities of proteins purified from mammalian cells were measured by BLI
Reconstitution of the interplay between actin and microtubules on micropatterns.Figure 5Schematic illustration of the micropatterning method to graft microtubules seeds (green) via neutravidin (yellow) and actin-nucleation-promoting complexes (orange) on 8-micron-wide discoidal micropatterns. A glass coverslip (deep blue) coated with polyethyleneglycol (PEG) (light blue) was placed in contact with a transparency mask and exposed to deep UV light. The exposed coverslip was then immersed with neutravidin to fix biotinylated microtubule seeds (green) on exposed regions. Streptavidin-WA was immobilized on microtubule seeds via their interaction with biotin. Tubulin dimers and actin monomers were then added to allow filaments elongation. Representative images of microtubules (top) and actin filaments (bottom) growth from micropatterns. Scale bars: 20 μm. Schematic illustration of the assay on micropatterned substrate. Tubulin was first added alone to measure the nucleation capacity of each micropattern, and then washed out. Later on, actin was added followed by actin and tubulin. Finally, actin was rinsed out and gelsolin was added to fully disassemble actin filaments. Representative images showing microtubules (top line) and the merged images of actin and microtubules (bottom line) during the four steps of the assay; in the presence of tubulin only, in the absence of tubulin and presence of actin, in the presence of tubulin and actin, and finally in the presence of tubulin and gelsolin but in the absence of actin filaments (from left to right). Scale bar: 10 μm. Quantification of the number of microtubules per micropattern in the presence of tubulin only (left), actin and tubulin (middle) and tubulin only after actin filaments disassembly (right). Data were pooled from 2 independent experiments; n= 133. ****:P<0.001 Student t-test. The graph shows the same measurements as in panel (E) in an XY representation of individual measurements. It illustrates the differences in the number of microtubules per micropattern between the first to the second step (tubulin only versus actin and tubulin together) with respect to the density of actin filaments per micropattern.
Parkin inhibits BAX-mediated apoptosis by indirect ubiquitination.Figure 1(A) Schematic of potential effects of activated Parkin on the BCL-2 family of apoptosis regulators. (B) BAX/BAK-deficient HeLa cells reconstituted with BAX and where indicated HA-Parkin were treated with antimycin A and oligomycin (AO) for 2 h and then 1 µM of each of the BH3 mimetics ABT-737 and S63485 for 6 h. Cell death was assessed at the indicated times by LDH assay. Data is mean +/- SD of four independent experiments. (C) BAX/BAK-deficient HeLa cells ectopically expressing BAX and Parkin were treated with antimycin A and oligomycin (AO, 3 h), MG132 (20 µM, 3.5 h) and BH3 mimetics (1 µM of each of ABT-737 and S63485, 1 h). Cytosol and membrane fractions were immunoblotted as indicated. Graph shows densitometric analysis of non-ubiquitinated membrane proteins (top, relative to untreated control) or cytochrome c release (bottom; cytosol/cytosol+membrane, relative to untreated controls) from three independent experiments. Error bars represent mean +/- SD. (D) BAX/BAK-deficient HeLa cells ectopically expressing BAX were treated and analysed (E) UBA pull-down of ubiquitinated proteins following antimycin A and oligomycin (AO) treatment of BAX/BAK-deficient HeLa cells ectopically expressing HA-Parkin and BAX for 3 h. Representative immunoblot from two independent experiments.
Fig.Figure 1A 3D structure of a HA trimer (PDB 3UBE generated with PyMol). Each monomer consists of a HA1 (dark grey) and HA2 (light grey) subunit. The trimer can be divided into a membrane distal head which contains the RBS (yellow) and a proximal stalk region. B HA and NP represent the Cal/7/9 wildtype proteins. Chimeric HA (cHA) 1 contains the head regions of H2 HA, cHA2 of H10 HA and cHA3 of H13 HA, while they all contain the Cal/7/9 HA-stalk region. Headless HA (HL) contains a deletion in the HA-head region (dashed line). Modified headless HA (mHL1 and mHL2) contain stabilizing mutations (black boxes) and lack additional internal parts. All constructs were codon-optimized and carry a V5-tag at their C-terminus. C Frequency of C179+ or V5-tag+ 293T cells 24h after transfection of AAV-vector plasmids as measured by flow cytometry. Symbols represent single experiments and bars the mean ± SE (n = 3). D Immunoblot of 293T cells 72 h after transduction with AAV-vectors at a MOI of 106. Antigen expression was detected with an anti-V5-tag antibody. Equal loading was controlled with a GAPDH antibody (n = 3).
Figure 1(A) ARPE-19 cells were incubated in the presence of DMSO (Control), Tunicamycin (5 µg/ml), or starved (EBSS) for 16 h. Cells were fixed, permeabilized and stained with an antibody against TFE3. Scale bar, 10 µm. Data are representative of three independent experiments. (B) Immunoblot of protein lysates of ARPE-19 cells treated as shown in (A). Data are representative of three independent experiments. (C) ARPE-19 cells incubated with Tunicamycin as indicated in (A) were immunostained with antibody against CHOP. Scale bar, 10 µm. Data are representative of three independent experiments. (D) Quantification of the percentage of ARPE-19 cells with nuclear TFE3 upon Tunicamycin (Tun.) or starvation (Strv.) treatments as indicated in (A) (mean ± SD of three independent experiments, one-way ANOVA analysis versus Control treated cells, ***p < 0.001; n>400 cells per condition). (E) Wild-type mouse embryonic fibroblast (MEF) cells were incubated in the presence of DMSO (Control) or Tunicamycin (0.1 µg/ml) for the indicated times. Cells were fixed, permeabilized and stained with antibodies against TFE3 (green) and CHOP (red). Scale bar, 10 µm. Data are representative of three independent experiments. (F) Wild-type MEF cells were incubated in the presence of DMSO (Control) or BFA (2.5 µg/ml) for 16 h. Cells were fixed, permeabilized and stained with antibodies against TFE3 (green) and CHOP (red). Scale bar, 10 µm. Data are representative of three independent experiments.(G) Quantification of the percentage of MEF cells with nuclear TFE3 upon DMSO (Control) or BFA 16 h treatment (mean ± SD of two independent experiments, student's t-test analysis versus DMSO treated cells, ***p < 0.001; n>400 cells per condition). (H) Immunoblots of the subcellular distribution of TFE3 and TFEB in MEF cells incubated with DMSO (Ctrl.) or Tunicamycin (Tun.) for 16 h using TFE3, TFEB, Lamp1 (as a lysosomal membrane marker), and H3b (as a nuclear marker). Data are representative of three independent experiments.
TBK1 and Gal8 are Required for the Recruitment of Autophagy Machinery to Early Endosomes.Figure 5The following experiments were performed in glial shNf-1/shTp53 glial cells serum starved 4 hr before assaying. Cells were expressing either Cas9 alone (control) or Cas9 and sgRNA targeting Atg7 (sgAtg7 #1 or #2). A: Cells were treated for 15 min with 2 ng/mL EGF before fixation and immunofluorescence staining against EEA1 and p-TBK1. White arrows indicate colocalisation. Scale bar: 10 μm. B: Quantification of percentage of total EEA1 vesicles that colocalise with p-TBK1 (in A). Data information: Statistical analyses were performed on at least 3 independent experiments, where error bars represent SEM and p values represent a two-tailed Student's t test: * p<0.05, ** p<0.01, *** p<0.001. The following experiments were performed in glial shNf-1/shTp53 glial cells serum starved 4 hr before assaying. Cells were expressing either Cas9 alone (control) or Cas9 and sgRNA targeting Atg7 (sgAtg7 #1 or #2). C: Cells stably expressing Flag-S-ATG16L1 were pre-treated for 1 hr with TBK1 inhibitors (100 μM MRT68601 or 5 μM momelotinib). Control cells were also treated 100 μM monensin for 1 hr as indicated. All cells were stimulated for 15 min with 2 ng/mL EGF followed by fixation and immunofluorescence staining against Flag tag and EEA1. Scale bar: 10 μm. The following experiments were performed in glial shNf-1/shTp53 glial cells serum starved 4 hr before assaying. Cells were expressing either Cas9 alone (control) or Cas9 and sgRNA targeting Atg7 (sgAtg7 #1 or #2). D: Quantification of total EEA1 vesicles that colocalise with Flag-S-ATG16L1 Data information: Statistical analyses were performed on at least 3 independent experiments, where error bars represent SEM and p values represent a two-tailed Student's t test: * p<0.05, ** p<0.01, *** p<0.001. The following experiments were performed in glial shNf-1/shTp53 glial cells serum starved 4 hr before assaying. Cells were expressing either Cas9 alone (control) or Cas9 and sgRNA targeting Atg7 (sgAtg7 #1 or #2). E: Western blotting of shNf-1/shTp53 glial cells expressing gRNA sequences targeting Gal8. The following experiments were performed in glial shNf-1/shTp53 glial cells serum starved 4 hr before assaying. Cells were expressing either Cas9 alone (control) or Cas9 and sgRNA targeting Atg7 (sgAtg7 #1 or #2). F: Control and sgGal8 cells stably expressing Flag-S-ATG16L1 were treated 100 μM monensin for 1 hr and stimulated with 2 ng/mL EGF for 15 min before fixation and immunofluorescence staining against EEA1 and Flag tag. White arrows indicate colocalisation. Scale bar: 10 μm. G: Quantification of percentage of total EEA1 vesicles that colocalise with Flag-S-ATG16L1 in sgGal8 cells (in F). Data information: Statistical analyses were performed on at least 3 independent experiments, where error bars represent SEM and p values represent a two-tailed Student's t test: * p<0.05, ** p<0.01, *** p<0.001.
S proteasome from human cytosolA.Figure 4A. Comparison of sequential digestion for complex mixtures with previous studies. Sequential digestion returns a higher number of residue pairs with low overlap to published datasets, showing the complementarity of the different approaches. Long distance links were determined only within proteins and are comparable for all datasets. B. Residue pairs for the TRiC/CCT complex were mapped into the crystal structure and support the rearrangement of the complex C. Despite the complexity of the sample we were able to identify the four states of the 26S proteasome showing the flexibility of the AAA-ATPase dependent heterohexameric ring.
Figure 3 (a) Representative micrographs of cluster formation assays conducted with the indicated proteins The total amount of ubiquitin moieties was kept identical in each condition (b) Quantification of the data shown in (a) (c) Quantification of cluster formation assays conducted with the indicated proteins. mCherry-p62 was pre-incubated with the indicated 4xUb variants, followed by the addition of streptavidin immediately before imaging (d) Representative micrographs of the indicated samples taken 60 minutes after addition of streptavidin (e) Quantification of cluster formation assays conducted with the indicated proteins (f) Quantification of cluster formation assays conducted with 2µM mCherry-p62 incubated with the indicated concentration of GST-GFP-4xUb. Box: the highest particle number per field detected in each sample was plotted against the relative GST-GFP-4xUb concentration. Data points were fitted to a sigmoidal curve. The regression coefficient is indicated (g) Quantification of cluster formation assays conducted with GFP-p62 and the indicated GST-tagged ubiquitin chains (h-i) Quantification of cluster formation assays performed with mCherry-p62 and GST-4xUb in presence of the indicated free ubiquitin chains. Ubiquitin chains were either pre-mixed with p62 before the addition of GST-4xUb (h), or added 3 minutes after the addition of GST-4xUb to p62 (i) (j) Quantification of cluster formation assays conducted with mCherry-2xFKBP-p62 in presence of AP20187, GST-4xUB and the indicated Ub chains (k) Representative micrographs of glutathione beads coated with the indicated GST-tagged proteins and incubated with the indicated mCherry-p62 variants. In all pictures the brightness was adjusted to highlight the profile of the beads. For all graphs averages and SDs from at least three independent replicates are shown
Figure 7(A-B) mRNA expression of human colonic stem cell markers LGR5 and OLFM4, n=5 or 6 independent human samples. Paired student t-test. (C) Representative images of LGR5 ISH in human organoids treated with DMSO or TCDD. Scale bar 50 µm. mRNA expression of target genes, n=5 or 6 independent human samples. Paired student t-test. mRNA expression of target genes, n=5 or 6 independent human samples. Paired student t-test. (H) Representative immunoblots for FoxM1 and PLK1 protein derived from organoids treated with DMSO or TCDD from n=6 independent samples. Total protein was used as a loading control. (I) Determination of luciferase reporter activity of human FoxM1 in SW48 and Caco2 cell lines treated with DMSO or TCDD. n=3-4 per group. (J) FOXM1 and CYP1A1 mRNA expression in different AhR WT and CRISPR KO clones treated with TCDD for 1 d. C56 and C61 designate different human subjects. (K) Validation of CRISPR KO clones was assessed by PCR amplification using primers flanking the targeted AhR exon. Subsequent sequencing revealed indels at the expected locations. (L) The association of FoxM1 and AhR mRNA expression in human normal colon biopsies (n=304). AhR low (n=152) is defined as below its median expression; AhR high (n=152) is defined as above its median expression. Dashed line indicates median and dotted lines indicate 25th/75th quartiles. (M) Correlation between FoxM1 and Cyp1b1 mRNA expression in human normal colon biopsies (n=303) analyzed by Spearman rank correlation. (N) Representative brightfield images of human colonic organoids from sorted live cells at day 6, scale bar 200 μm. (O, P) (O) Human organoid forming efficiency, and (P) organoid diameter from sorted individual cells from human colonic organoids in the presence of DMSO, TCDD and CH223191 (10 μM) for 6 d. n=5 per group, 3 independent experiments. Grey line indicates mean in (P). Treatments not sharing a common letter are significantly different, p<0.05. (Q) Representative brightfield images of human colonic organoids treated with DMSO or FDI-6. FoxM1 inhibition suppressed human colonic organoid growth in a dose-dependent manner, scale bar 200 µm.
Figure 6(A) The number of receptor SSDs, segmented from the combined receptor clusters of GABAARs and GlyRs, was decreased after 4-AP treatment (MW test, p < 0.0001). (B) This was mostly due to the loss of GABAAR SSDs that were segmented from the rendered images of GABAAR detections only (MW, ****p < 0.0001). (C) The number of GlyR SSDs (based on GlyR detections) were only marginally reduced (MW, **p < 0.01). Data information: Number of synapses: n = 452 in TTX, n = 583 in 4-AP condition, from three independent experiments. Mean ± SEM.
TGF-α regulates the Hippo signaling pathway in cervical cancer cells.Figure 6Western blot analysis showing effects of TGF-α on phosphorylation of EGFR, YAP, AKT, and ERK1/2. ME180 cells were starved for 6 h after reaching cell confluence, then cells were treated with TGF-α (100 ng/ml) for 0, 5, 10, 30, 60 or 120 min. Western blot analysis showing effects of TGF-α on the phosphorylation of the components of the Hippo signaling pathway. ME180 cells were starved for 6 h after reaching cell confluence, then cells were treated with TGF-α (100 ng/ml) for 0, 30, 60 min. Real-time PCR determines the action of TGF-α and YAP levels on the mRNA expression of AREG. Each bar represents the mean ± SEM (n = 5). Bars with different letters are significantly different from each other (Ctrl vs. TGF-α, P < 0.0001; MXIV vs. YAP, P < 0.0001; MXIV vs. YAPS127A, P < 0.0001). Real-time PCR analysis showing that knockdown of YAP with YAP siRNA (siYAP) significantly suppressed TGF-α-induced (FBS 10%, TGF-α: 10 ng/ml for 48 h) AREG expression in ME180 cells. siCTRL, a non-targeting siRNA, was used as a negative control. Each bar represents the mean ± SEM (n = 5). Bars with different letters are significantly different from each other (siCTRL vs. siCTRL+TGF-α, P < 0.0001; siYAP+CTRL vs. siYAP+TGF-α, P < 0.0001; siCTRL+TGF-α vs. siYAP+TGF-α, P = 0.0067). Proliferation of ME180 cells (FBS 1%) treated with control (siCTRL) or YAP (siYAP) prior to treatment with control medium or TGF-α (10 ng/ml) for 108 h. Each bar represents the mean ± SEM (n = 4). Bars with different letters are significantly different from each other (siCtrl vs. siCTRL+TGF-α, P = 0.0058; siYAP+CTRL vs. siYAP+TGF-α, P = 0.1840; siCTRL+TGF-α vs. siYAP+TGF-α, P = 0.0013). Quantitative data of the wound-healing assay showing the migration of ME180 cells that were treated with control (siCTRL) or YAP siRNA (siYAP) prior to treatment with or without TGF-α for 12 h. Each bar in bar graphs represents the mean ± SEM (n = 4). Bars with different letters are significantly different from each other (siCtrl vs. siCTRL+TGF-α, P < 0.0001; siYAP+CTRL vs. siYAP+TGF-α, P < 0.0001; siCTRL+TGF-α vs. siYAP+TGF-α, P = 0.0041).
Rab11-FIP3 regulates T cell spreading and cortical rigidity independently of TCR engagement.Figure 5A, B, C: JurkatT cells transfected with siRNA control, FIP3.1 or FIP3.2 were allowed to spread on poly-lysine-coated coverslips for the indicated times. Intracellular actin was detected with FITC-coupled Phalloidin. A: A single optical section at the level of the contact surface is shown. Bar, 5 µm. B: Population analyses of the spreading area of cells at the level of the contact surface. Each dot represents one cell. Horizontal bars represent the mean ± SEM. C:Time course representation of B, showing mean ± SEM. D-F: Primary humanCD4T cells were transfected with control or FIP3.1 siRNA. D, E: Cells were allowed to spread on poly-lysine-coated coverslips for 5 min. Intracellular Lck was detected by immunofluoresecence. D: A single optical section at the level of the contact surface is shown. Bar, 10 µm. E: Population analyses of the spreading area of cells at the level of the contact surface. F: Cells were stained with CMFDA dye, suspended in a paraformaldehyde solution and submitted to centrifugation at 3,724 xg onto poly-lysine-coated coverslips. Confocal optical sections separated by 0.2 µm were acquired. X-Z images were built with ImageJ. The maximal sections in 'x' (width) and 'z' (thickness) were determined for each cell (upper panel). Plots represent the deformability index (x/z). Each dot corresponds to one cell. Horizontal bars represent the mean ± SEM. Mann-Whitney test. Images are representative of three experiments. G: FIP3 expression was analyzed by Western blot. Quantification of bands was performed with ImageJ and normalized to a non-specific band of 35 kDa used as loading control.
Figure 5(A) Immunoblots of protein lysates from ARPE-19 cells infected with either adenovirus expressing Null, TFEB-S211A, or TFE3 upon incubation with DMSO (Control), Tunicamycin (5 µg/ml) or starved in EBSS for 16 h. Data are representative of five independent experiments.(B) Quantification of ATF4, CHOP and GADD34 protein levels in ARPE-19 cells treated as indicated in (A) (mean ± SD of the fold change of the indicated protein to actin ratio from three independent experiments, one-way ANOVA analysis versus the corresponding treatment condition in the adenovirus-Null infected cells, *p < 0.05, **p < 0.01, ***p < 0.001). (C) Relative Quantitative Real-Time PCR analysis of ATF4, CHOP, GADD34 and MCOLN1 mRNA transcript levels in ARPE-19 cells treated as indicated in (A) (mean ± SD of the RNA fold change of indicated genes normalized to actin mRNA from three independent experiments, one-way ANOVA analysis versus the corresponding treatment condition in the adenovirus-Null infected cells, **p < 0.01, ***p < 0.001).