EMBO/sd-character-level-ner huggingface dataset:
Figure 4A-I. Dye release time courses from liposomes as a percentage of maximal release. A. Five different reactions, where 5 nM caspase-1 and 400 μM 6-carboxifluorescein-loaded liposomes prepared with E. Coli polar lipid extract were incubated with GSDMD concentrations of (nM): 520, 260, 130, 100, 65 (colored dark to light orange). The time point of 20 min is highlighted by a vertical dashed line. A-I. Dye release time courses from liposomes as a percentage of maximal release. B. Five different reactions, where 130 nM of GSDMD and 400μM 6-carboxifluorescein-loaded liposomes prepared with E. Coli extract polar lipid were incubated with caspase-1 concentration of (nM): 15, 8, 5, 2.5, 1.2 (colored dark to light blue). The time point of 20 min is highlighted by a vertical dashed line. A-I. Dye release time courses from liposomes as a percentage of maximal release. C. Three different reactions, where 5 nM caspase-1 and 400 μM 6-carboxifluorescein-loaded liposomes prepared with porcine brain total lipid extract were incubated with GSDMD concentrations of (nM): 520, 260,100 (colored dark to light green). A-I. Dye release time courses from liposomes as a percentage of maximal release. D. Dye release at 20 minutes reaction as a function of GSDMD (dark orange) and caspase-1 (dark blue) concentrations. Errors bars for three independent experiments are shown. A-I. Dye release time courses from liposomes as a percentage of maximal release. E. Two different sets of reactions, where wild type GSDMD (dark to light orange) and the mutant GSDMDI104N (dark to light blue) were independently incubated at the concentrations of 260, 130 and 65 nM with 5 nM caspase-1 and 400 μM 6-carboxifluorescein-loaded liposomes. The time point of 60 minutes is highlighted by a vertical dashed line. A-I. Dye release time courses from liposomes as a percentage of maximal release. F. Dye release at 60 minutes of reaction as a function of GSDMD wild type (dark orange) and GSDMDI104N (dark blue) concentration. Error bars for three independent experiments are shown. A-I. Dye release time courses from liposomes as a percentage of maximal release. G-I. Dye release from 400 μM liposomes loaded with the 6-carboxifluorescein-derivates FD-20, FD-40 and FD-150, with variable Stokes diameters, as indicated. 130 nM of GSDMD and 5 nM caspase-1 were incubated with the liposomes.
Figure 4SUD and PABP do not compete with each other for interacting with Paip1. (A) Excess expression of SUD-RFP does not impair the split-YFP signal between PABP and Paip1 (lower images), compared to the control (upper images). (B) Co-expression of excess RFP-PABP does not result in an impaired interaction between Paip1 and SUD either. RFP: red fluorescent protein. Scale bars represent 50 µM. GFP-trap-based co-immunoprecipitation (CoIP) assay demonstrating a complex formed by PABP, Paip1, and SUD. GFP: green fluorescent protein.
Figure 4Imaging of SPBs (Cnm67-RFP) and Spc72-GFP or Mpc70-GFP in cells containing different SPO13 alleles and/or PEST-clb1-mDK, which was induced with estradiol at 4 h in SPM. (A) Representative time-lapse series. (B) Analysis of cells containing wild-type Spo13, Spo13-mD, or Spo13-mD-m2. Meiotic events were quantified in cells synchronized in silico to SPB reduplication at entry into metaphase II (t = 0). Graphs show overlays of SPC72-GFP and MPC70-GFP strains. spo13-mD delays Spc72 removal and Mpc70 loading by 30 min (95% CI, 26-34; P < 0.0001) and 60 min (95% CI, 55-66; P < 0.0001; Welch's t-test), respectively. (C) Analysis of cells expressing Clb1-mDK and different versions of Spo13. Meiotic events were quantified as in (B). spo13-mD plus PEST-clb1-mDK delays Spc72 removal and Mpc70 loading by 72 min (95% CI, 63-80; P < 0.0001) and 114 min (95% CI, 99-129; P < 0.0001; Welch's t-test), respectively. (D) Time from SPB reduplication at metaphase II to Mpc70 loading plotted versus time from SPB reduplication to Spc72 removal for strains with the indicated genotypes. Diamonds represent mean values, and error bars indicate data range. Numbers of independent experiments used to calculate mean values are shown on the right.
Figure 6The amino acid sequences surrounding confidently localised sites of (A) pHis, (B) pAsp, and (C) pGlu (ptmRS ≥ 0.99) were analysed for sequence enrichment using Motif-X. Depicted are the sequences of the enriched motifs. Additional details are presented in Appendix Figs. S8, S11, S13).
Figure 2A-B. Cleaved caspase-3 positive muscle fibers (*).A. PEO patient (P3) on normal diet (inset: caspase staining in healthy control individual)B. PEO patient (P3) after modified Atkins diet. Arrowhead: caspase negative necrotic fiber. C. Western blot analysis of autophagy receptor p62 and pax7 marking muscle satellite cell activation; left, blot; right, signal quantification in each patient. D - E. Immunohistochemistry analysis of p62 and mitochondrial mass marker, consecutive sections. Asterisks indicate p62-positive fibers and corresponding affected ragged-red fibers.F - G. p62 and mitochondrial mass after mAD; consecutive sections. Arrowheads indicate fibers that show p62 positivity but have normal mitochondrial mass. H. p62 and LC3 positive ragged-red fibers (*) in PEO patient (P2), normal diet.I. Muscle fibers showing granular LC3-positivity, PEO patient (P1), after mAD. Immunohistochemistry on frozen sections; bars: 50 µm, except for H, 100 µm.
Figure 2(A) The PSR induces the cleavage of PRORALF23-GFP into mature RALF23-GFP. The 35S promoter-driven RALF23-GFP line was grown in either HP or LP media for 5 days and roots were harvested. Premature and mature RALF23 levels were determined with anti-GFP. β-actin was used as a loading control. Data information: All experiments were repeated three times with similar results. (B) The PSR activates RALF expression under LP conditions. Col-0 seedlings were grown in either HP or LP media for 5 days and roots were harvested. The relative expression of RALFs was quantified via RT-qPCR. The data shown indicate the means ± SDs (n = 3, n refers to technical replicates); n.s., not significant; *, p < 0.05; **, p < 0.01 (Student's t test). Data information: All experiments were repeated three times with similar results. (C) Expression pattern of RALFs in the shoots and roots under Pi-starvation conditions. Data information: All experiments were repeated three times with similar results. (D) PHR1-binding site (P1BS, GNATATNC) prediction within 1000 bp of certain RALF promoters. The positions are relative to the start codon (+1). Data information: All experiments were repeated three times with similar results. (E) The PSR activates RALF expression in a PHR1-dependent manner. Col-0 and phr1 seedlings were grown in LP media for 5 days and roots were harvested. The relative expression of RALFs was quantified via RT-qPCR. The data shown indicate the means ± SDs (n = 3, n refers to technical replicates); n.s., not significant; **, p < 0.01 (Student's t test). Data information: All experiments were repeated three times with similar results. (F) PHR1 activates RALF promoters in plants. Depicted RALF promoter-LUC fusions (RALF24 as a negative control) were co-expressed together with 35S promoter-driven PHR1 or an empty vector control in Arabidopsis protoplasts. The relative LUC/REN reporter activity was quantified. The data shown indicate the means ± SDs (n = 3, n refers to technical replicates); n.s., not significant; *, p < 0.05; **, p < 0.01 (Student's t test). Data information: All experiments were repeated three times with similar results. (G) PHR1 directly binds to RALF promoters, as shown via ChIP-PCR. The Myc-labelled PHR1 overexpression line was immunoprecipitated with anti-Myc. ChIP analysis was performed on Col-0, CRY1-Myc and PHR1-Myc transformed plants, with Col-0 and CRY1-Myc transformed plants as negative controls. The data shown indicate the means ± SDs (n = 3, n refers to technical replicates); n.s., not significant; **, p < 0.01 (Student's t test). Data information: All experiments were repeated three times with similar results.
Figure 2(A) Fluorescence time-lapse images of F-actin, plasma membrane and histone during QR.ap migration in mig-2(rh17) gain-of-function mutant animals. Merged images, left; inverted fluorescence images of F-actin, right; white arrows, migration direction; asterisks, nucleus; double headed arrow, migration distance. The dotted lines indicate the cell periphery. Time is presented in minutes. Scale bar, 5 μm. (B - C) Quantification of the QR.ap migration angle (B) and distance (C). N = 10. (D) Mass spectrum of a MIG-2 peptide that was phosphorylated at Serine 139 in C. elegans. The peptide was identified from the GFP::MIG-2 immunoprecipitate. (E) In vitro phosphorylation assay of CST-1/2 to MIG-2. Bacterially expressed His-tagged MIG-2 or His-tagged MIG-2S139A were incubated with the GST-tagged kinase domain of GST-CST-1/2 (1-318 aa). The kinase may use ATP-γ-S as a phosphodonor to thiophosphorylate its substrate; thus, the phosphorylation reaction was performed with ATP-γ-S, and anti-thiophosphate ester antibody was used to detect substrate phosphorylation (Allen et al, 2005; Allen et al, 2007). The final reaction was probed via immunoblotting with antibodies as indicated. Thio, thiophosphate ester. (F) Sequence alignment of MIG-2. Serine 139 (red) is conserved among the Rac1 homologs across species. The accession version is as follows: C. elegans, CAB01691.1; Drosophila, CAC88352.1, AAA67041.1; Human, CAB53579.5; Rat, NP_599193.1; Mouse, AAH51053.1; Chicken, NP_990348.1, NP_001188381.1; Zebrafish, AAV85902.1; Xenopus, NP_001089332.1. (G H) Three-dimensional ribbon (PDB 3TH5) representations of the human Rac1 polypeptide which indicate the position of the T135 (S139 in MIG-2) located between the last two α-helixes (Krauthammer et al, 2012).
Figure 1A. Retinal whole mount immunostaining for Isolectin-B4 (red) and Desmin (green) in WT (top panel) and Glut1iECKO (bottom panel) mice showing pericyte coverage in the vasculature. Left and right panels show representative images with 20x or 63x magnification, respectively. B. Quantification of the pericytes coverage in the retinal vasculature of WT (blue) and Glut1iECKO (orange) mice. (n=9 from 3 independent experiments. Error bars indicate the standard error of the mean (SEM) from unpaired Student's t test.) C. Immunostaining for CD31 (green), CD13 (white; pericyte marker) and αSMA (cyan) using paraffin section from WT (top panel) and Glut1iECKO (bottom panel) mice. D. Quantification of the pericytes coverage using CD13 immunostaining in the brain vasculature of WT (blue) and Glut1iECKO (orange) mice. (n=7 from 3 independent experiments. Error bars indicate the standard error of the mean (SEM) from unpaired Student's t test.) E. Quantification of the smooth muscle cell coverage using αSMA immunostaining in the brain vasculature of WT (blue) and Glut1iECKO (orange) mice. (n=13 from 3 independent experiments. Error bars indicate the standard error of the mean (SEM) from unpaired Student's t test.) F. Immunostaining for Cldn5 (green) and CD31 (red) using a brain section of WT (top panel) and Glut1iECKO (bottom panel) mice. G. Immunostaining for caspas-3 (green), CD13 (red) and CD31 (cyan) using a brain section of WT (top panel) and Glut1iECKO (bottom panel) mice. H. Permeability assay using vibratome section showing the leakage of retro-orbital injected cadaverine dye (red) in the brain of WT (top panel) and Glut1iECKO (bottom panel) mice. DAPI staining (white) shows the boundary of brain section. I. Permeability assay using vibratome section showing the leakage of retro-orbital injected cadaverine dye (red) in the brain of WT (top panel) and Glut1iECKO (bottom panel) mice. Note that cadaverine is retained in the intravascular area of WT mice (upper panel), but extravasated and accumulated in the brain parenchyma (white arrowheads) of Glut1iECKO (bottom panel) mice. J. Representative confocal images of fibrin (green), CD31 (red) and DAPI (blue) immunostaining in the brain vasculature of WT (top panel) and Glut1iECKO (bottom panel) mice K. Quantification of the number of cadaverine 555 positive brain parenchyma cells in WT (blue) and Glut1iECKO (orange) mice. (n=13 from 3 independent experiments. Error bars indicate the standard error of the mean (SEM) from unpaired Student's t test.) L. Quantification of extravascular fibrin deposits in WT (blue) and Glut1iECKO (orange) mice. (n=9 from 3 independent experiments. Error bars indicate the standard error of the mean (SEM) from unpaired Student's t test.) M. Quantification of lactate in the cerebrospinal fluid of WT (blue) and Glut1iECKO (orange) mice. (n=3 from 3 independent experiments. Error bars indicate the standard error of the mean (SEM) from unpaired Student's t test.)
Figure 6A. In left, normalized peak amplitudes of isolated AMPA EPSCs recorded at -60 mV, before and after pairing protocol, are illustrated. Representative traces of AMPA EPSCs before (1) or after (2) the pairing protocol are also illustrated (right panel) for each experimental condition: Vehicle-injected mice (white circles, n=3 from N=3), THC-injected mice (green circles, n=3 from N= 3) and THC-injected mice treated with SB258585 during adolescence (black circles, n=3 from N=3). Errors bars correspond to mean ± SEM. B. The histogram represents the means ± SEM of AMPA EPSCs in % of baseline, measured during the last 5 min of baseline or the last 5 min of recording, for each experimental condition: vehicle-injected mice (white bar, n=3 from N=3), THC-injected mice (green bar, n=3 from N= 3) and THC-injected mice treated with SB258585 during adolescence (black bar, n=3 from N=3), n.s. p > 0.05, * p< 0.05, one-way ANOVA followed by Tukey test.
Figure 1(A) A431 WT and SKO cells -/+ GFP-fused WT or A212P-seipin were delipidated, treated with OA for 1 h, fixed and stained with LD540. Maximum intensity projections of deconvolved confocal z-stacks. Dashed lines indicate cell boundaries. (B) Analysis of LD areas of cells treated as in (A) and imaged with widefield microscopy. Bars: % of LDs/cell in indicated size category, mean ± SEM, n=57-61 cells/genotype, 2 experiments, p**< 0.005 (unpaired T-test). (C) SKO + WT- or A212P-seipin-GFP cells were stained for calreticulin. Maximum intensity projections of deconvolved confocal z-stacks. (D) SKO + WT- or A212P-seipin-GFP cells were grown in standard growth medium, fixed and stained with anti-GFP antibodies. Widefield and STORM images. (E) SKO + WT-seipin-GFP cells were delipidated and treated with OA for 1h, fixed and stained with LipidTox Red. Single focal plane of a 3D-SIM z-stack. (F) Analysis of (E), bars: % of LDs/cell with the indicated number of WT-seipin-GFP punctae associated, mean ± SEM, n=12 cells (693 LDs), 2 experiments, p**< 0.005 (Tukey multiple comparisons test). (G) SKO + WT- or A212P-seipin-GFP cells were delipidated, incubated with OA for 30 min, and immunolabelled for TEM with anti-GFP antibodies. Black arrowheads: immuno-gold, orange arrowheads: ER-LD junctions. See also Appendix Fig S3. (H) Analysis of (G), bars: relative labeling index in different cellular compartments, n=3456-4512 immuno-gold clusters. Distribution of both WT- and A212P-GFP-seipin immunolabel is non-random (chi square test, **p<0.005), but there is significant enrichment of WT- but not A212P-seipin-GFP at ER-LD contacts relative to the ER (binomial test, **p<0.005). See also Appendix Table S1 and S2. (I) A431 WT cells with seipin tagged with sf-GFP at its chromosomal locus and stably expressing ER marker plasmid (BFP-KDEL) were delipidated and treated with OA for 1 h, fixed and stained with LipidTox Red. Single confocal plane of an airyscan z-stack.
Figure 4(A) Triple immuno-labelling of gephyrin, GlyRs and GABAARs at mixed inhibitory synapses (arrow) imaged with conventional fluorescence microscopy. Scale bar: 5 µm. (B) Quantification of the apparent synaptic area (in pixels) of gephyrin clusters (KS test p < 0.0001). (C) Lower fluorescence intensity of GABAARs but only a minor reduction of GlyRs at synaptic gephyrin clusters (KS test, p < 0.0001, both panels). (D) Reduced GABAAR/GlyR intensity ratio at mixed inhibitory synapses (KS test, p < 0.0001). Number of synapses: n = 9416 in TTX and n = 6949 in 4-AP conditions from three independent experiments.
UVRAG interacts with Bax.Figure 4(A,B) HL60 and HCT116 cells were treated with UV irradiation for 24 h and then assayed for protein expression levels as indicated by co‐immunoprecipitation (RIPA or CHAPS buffer) or western blotting. (C) Proteins in the pellet after GST pull‐down assays in HCT116 cell lysate were analysed by SDS-PAGE and immunodetected with UVRAG or GST antibodies. (D) UVRAG C2 domain deletion terminates BAX binding. HCT116 cells were transfected with Flag‐UVRAG or its mutants (ΔC2, ΔCCD), and then assayed for protein expression levels as indicated by IP or western blot. (E) Apoptosis was assayed in indicated HCT116 cells after treatment with doxorubicin (Doxo, 1 μg/ml), UV irradiation (5 min after 50 mJ/cm2) or transfected with BAX (1 μg) for 24 h. BAX, BCL2‐associated X protein; CCD, coiled‐coil domain; CHAPS, 3‐[(3‐cholamidopropyl)‐dimethylammonio]‐1‐propane sulphonate; GST, glutathione‐S‐transferase; IB, immunoblotting; IgG, immunoglobulin G; IP, immunoprecipitation; RIPA, radioimmunoprecipitation assay; SDS-PAGE, SDS-polyacrylamide gel electrophoresis; UV, ultraviolet; UVRAG, ultraviolet irradiation resistance associated gene.
Figure 3(A) The protein domain structure of RDH/CG2064. Grey box: predicted transmembrane region; Pink box: short-chain dehydrogenase domain. The key enzymatic motif (YXXXK) and active site (NXXG) are shown. (B) 3rd instar larval fat bodies were stained by BODIPY (green) for lipid droplets and DAPI (blue) for nuclei. Overexpressing wild-type or site-mutated RDH/CG2064 increases the lipid droplet size in larvae with DGAT1 overexpression (ppl>DGAT1). Scale bar represents 25 µm. (C) Quantification of the lipid droplet size in (B). (D) Immunofluorescent staining of RDH/CG2064-GFP with anti-GFP antibody in 3rd instar larval fat body. DAPI (blue) stains nuclei. RDH/CG2064 is enriched in the peri-nuclear region. Scale bar represents 25 µm. (E) Immunofluorescence imaging of RDH/CG2064-GFP, detected with anti-GFP antibody, and the ER protein marker RFP-KDEL in 3rd instar larval fat body. RDH/CG2064 co-localizes with RFP-KDEL. Scale bar represents 10 µm. (F) Whole lysates and cytosolic and membranous fractions were prepared from fat body samples of 3rd instar larvae expressing an RDH/CG2064-GFP knockin reporter. RDH/CG2064-GFP protein was detected by anti-GFP antibody in a western blot. RDH/CG2064-GFP is mainly present in the membranous fraction.
Cytoskeleton remodelling in B lymphocytes upon antigen stimulation.Figure 1IIA1.6 B lymphoma cells were stimulated with BCR-ligand- (IgM) or BCR-ligand+ (IgG) beads for 60 min, fixed and stained for F-actin (top) and α-tubulin (bottom). Scale bar: 3 µm. Histograms show the quantifications of the polymerized tubulin and filamentous F-actin at the centrosome (dashed outline on the image, values correspond to the fraction of fluorescence in a 2-micron-wide area around the centrosome relative to the total fluorescence in the cell) and the total amount of polymerized fluorescent tubulin (bottom right, values were normalized with respect to the mean of control condition). Measurements were pooled from 3 independent experiments; IgM (BCR-ligand-): n= 88; IgG (BCR-ligand+): n= 93. Error bars correspond to standard deviations. P values were calculated with Mann-Whitney test. Scale bar: 2 µm. Percentage differences of centrosomal F-actin and centrosomal microtubule fluorescence intensities in cells stimulated with BCR-ligand+ beads with respect to cells stimulated with BCR-ligand- beads. The data set is identical to panel (B). Measurements were pooled from 3 independent experiments; IgM (BCR-ligand-): n= 88; IgG (BCR-ligand+): n= 93. Error bars correspond to standard deviations. P values were calculated with one-sample t-test (i.e. comparison to a theoretical mean of "0"). The graph shows the variations of the total amount of tubulin per cell with respect to the content of actin at the centrosome in an XY representation of individual measurements. The two lines correspond to linear regressions of the two sets of data relative to cells stimulated with BCR-ligand+ (activated cells) or BCR-ligand- (resting cells) beads. IIA1.6 B lymphoma cells were fixed and immuno-stained for F-actin (red) and microtubules (green). Images show the projection of maximal intensity of three confocal slices spaced by 0.5 µm apart from the centrosome. Scale bars: 2 µm (0.5 µm in the zoomed insets).
Suprabasal BSCs are prone to detachment-induced apoptosis.Figure 3A GO classification of differentially regulated genes in P50 suprabasal BSCs (CD34+/Itgα6low) compared to basal BSCs (CD34+/Itgα6high) clustered for biological process (clustering and statistical analysis done with Metascape, according to Zhou et al. 2019 (Zhou et al, 2019). Columns of interest in red. B Functional classification of selected differentially expressed genes in sorted suprabasal BSC vs basal BSC after RNA-seq analysis (red: up-regulated genes, green: downregulated genes; fold change > 2, p < 0,05 C qRT-PCR for mRNA expression of the anoikis-related markers Bim and Bmf in sorted n-B (non-bulge), bBSC (basal BSCs) and sbBSC (suprabasal BSCs) (n=3 biological replicates; mean ± standard error of mean (SEM); * p<0.05, ns=not significant, two-way ANOVA test).
Acetylation at K274 and K279 disrupts the dimerization and DNA binding of TFEB.Figure 5A Subcellular localization of TFEB in WT or GCN5 KO HEK293 cells treated with or without Torin1 (Scale bars, 10 µm). B Subcellular localization of Flag-tagged TFEB or TFEB mutants in HEK293 cells treated with or without Torin1. Cells were stained with anti-Flag antibody (Scale bars, 10 µm). C ChIP-qPCR analysis of TFEB binding to the promoter of its target genes CLCN7, GLA and CTSD. Normal mouse IgG and primers against the upstream region lacking CLEAR sites (up) were used as negative controls (mean ± SEM; n = 3 independent experiments; *p < 0.05, **p < 0.01, ***p < 0.001, Student's t-test). D Electrophoresis mobility shift assay of TFEB binding to the promoter of GLA. A DNA fragment from the GLA promoter containing the TFEB binding site was incubated with purified recombinant TFEB or each of the TFEB mutants, and was subjected to electrophoresis. Asterisk indicates the DNA fragment from the GAPDH promoter which was used as a TFEB non-binding DNA control. E Molecular dynamics snapshot of homodimer formation by human MITF and MITF mutants. K243 and K248 in the bHLH-Zip domain of human MITF correspond to K274 and K279 in human TFEB respectively. Steric interference is shown by yellow lines. Images were created using Chimera and Protein Data Bank (PDB) accession 4ATH. F Co-precipitation of Flag-tagged TFEB and TFEB mutants with Myc-TFEB in HEK293T cells. Immunoprecipitation was carried out with anti-Myc beads, and the precipitates were analyzed using anti-Flag. Co-precipitation of Flag-tagged TFEB and TFEB mutants with Myc-tagged MITF (G) in HEK293T cells. Co-precipitation of Flag-tagged TFEB and TFEB mutants with Myc-tagged TFE3 (H) in HEK293T cells. I TFEB homodimer formation detected by glutaraldehyde (GA) crosslinking. Purified recombinant GST-tagged TFEB or TFEB mutants was incubated with or without glutaraldehyde, then the products were analyzed by immunoblotting using anti-TFEB.