EMBO/sd-character-level-ner huggingface dataset:
Figure 3Hinge 2 (emerald) residues interacting with the ribosome. R565E forms a conserved cation-π-stacking with A329 of h8, R574 forms a salt-bridge with the phosphate backbone of U328 in h14. Aromatic C-terminal residues Y592 and Y593 adopt a parallel coordination. R572 of hinge 2 and N305 of hinge 1 (light green) form an interaction that might be important for sensing. Essential S580 does not contact the ribosome, whereas Y581 and E588 form H-bonds to R69 and M1 of eS6 (blue), respectively. Mutations in the α-helices of hinge 2 prevent 30S binding while the Y592A/Y593A (C-terminus) and L353Y (A-loop in NBSII) exchanges do not influence ribosome binding. 70S splitting efficiency normalized to wildtype. Hinge 2 mutations Y592A/Y593A, R565E and S580E display strongly impaired splitting activity. Unspecific ribosome dissociation level as determined in control experiments in the absence of ABCE1 is marked by the dotted line. ATP turnover per ABCE1 is not affected in all tested mutants. Overview of ATP coordination in both NBSs and overlay of the two NBSs reveals only slight differences, which cannot elucidate the functional asymmetry. Residues of NBD1 and NBD2 involved in coordination are shown in gold and punch, respectively.
Figure 5(A) Experimental design of TCR sequencing. (B) Percent T cells of estimated cells in BrM and CLN of mice from control or WBRT group. (C) Maximal productive frequency of clones in BrM and CLN samples. (D) Productive clonality in BrM and CLN samples. (E) Clonal space homeostasis of BrM samples indicating the relative abundance of clones with a specific frequency. (F) Lorenz curves generated with the reads of all clones in a sample to visualize deviation from perfect equality. (G) Lorenz curves were used to determine the Gini index of each sample, which is a measure of inequality reflecting clonal expansion. (H) Percent of estimated T cells correlated to BrM volume. (r=-0.7319, p=0.0252) (I) Gini indices of BrM samples correlated to BrM volume (r=-0.9453, p=0.0001).
/MD-2 induces receptor oligomerization.Figure 8(A, B) Surface electrostatic potentials of reported crystal structure of human TLR4/MD-2/LPS complex (3FXI), and mapping of putative GM3-binding pocket (A). Candidate basic residues and a hydrophobic pocket recognizing sialic acid and ceramide structure of GM3 are indicated (schematic) (B). (C, D) Alanine scanning for basic residues involved in signal transduction via VLCFA-GM3 (n=5) (C), and combinations of effective mutations (n=6) (D). Signal transduction was monitored by NF-κB reporter assay. (E-G) Comparative inhibitory effects of GM3 16:0 on mTLR4/mMD-2 (E), mTLR4/hMD-2 (domain-swapped complex) (F), and hTLR4/hMD-2 (G) (n=3). (H) Cross-linked SDS-PAGE analysis of recombinant mTLR4 (extracellular domain)/mMD-2 complexed with GM3 18:0, GM3 species enhancing mTLR4 activation.
Pharmacological inhibition of USP28 with the first-generation inhibitor AZ1 regulates ΔNp63 stability via de-ubiquitylation and shows a selective antiproliferative response of SCC cells Immunoblot of endogenous USP28 in A-431 cells upon treatment with warheads (WH) and either DMSO or indicated concentrations of AZ1 (USP28 Inh.Figure 7Immunoblot of endogenous USP28 in A-431 cells upon treatment with warheads (WH) and either DMSO or indicated concentrations of AZ1 (USP28 Inh.) for 24 hours. USP28 Upper band: Active USP28; USP28 lower band: Inactive USP28, VINCULIN as loading control. Immunoblot of endogenous USP28, ∆Np63 and KRT14 in A-431 cells treated for 24 hours with either DMSO or indicated concentrations of AZ1. VINCULIN served as loading control. TP63 and USP28 half maximal inhibitory protein abundance (IC50) was calculated. Graphs are represented as mean ± SD (n=3). TUBE pulldown of endogenous ubiquitylated ∆Np63 in A-431 cells upon treatment with either DMSO or indicated concentrations of AZ1 for 24 hours. VINCULIN served as loading control for relative Ubiquitination. Immunoblot of endogenous USP28, ∆Np63 and KRT14 in A-431 cells treated for 18 hours with either DMSO or 15µM AZ1 followed by 6 hours of treatment with 20µM proteasomal inhibitor MG-132 (20µM) or DMSO. VINCULIN served as loading control. A-431 cells were seeded at equal cell density and cultured in the presence of either DMSO, 1µM, 10µM or 30µM AZ1 for 48h. Cells were quantified with the Operetta imaging system using Hoechst staining. 50% growth inhibition (GI50) was calculated. Scale bar = 250µm. n= 30 fields analysed Immunoblot of endogenous USP28, ∆Np63 and KRT14 in LUDLU-1adh, H1299, Ca Ski, Hela and SiHa cells treated for 24 hours with either DMSO or indicated concentrations of AZ1. VINCULIN served as loading control LUDLU-1ADH, H1299, Ca Ski, Hela and SiHa cells were seeded at equal cell density and cultured in the presence of either DMSO, 1µM, 10µM or 30µM AZ1 for 48h. Scale bar for LUDLU-1adh and H1299 = 500µm. Scale bar for Ca Ski, Hela and SiHa = 250µm. SiHa* = Notably, the human SCC cell line SiHa was negative for ΔNp63. Number of cells were quantified with the Operetta imaging system using Hoechst staining. 50% growth inhibition (GI50) was calculated. n=30 fields analysed
VEGF impairs vascular stabilization despite similar pericyte coverage.Figure 1A Immunofluorescence staining of endothelium (CD31, in red), pericytes (NG2, in green), smooth muscle cells (α-SMA, in cyan), and nuclei (DAPI, in blue) on frozen sections of limbmuscles injected with myoblast clones expressing different VEGF levels (V Low, V Med, and V High, respectively) and treated with VEGF-Trap or saline after 10 and 17 days. Analysis was performed after 4 days of treatment (2 and 3 weeks, respectively). All normal vessels induced by low and medium VEGF doses displayed a similar coverage by normal pericytes, whereas aberrant structures induced by high VEGF were covered with α-SMA-positive smooth muscle cells. Asterisks indicate muscle fibers, which were mostly sectioned in longitudinal orientation, although some cross sections are visible depending on the orientation of muscle bundles. Scale bar = 25 μm.B, C Quantification of vessel length density (VLD) on the same samples showed that stabilization started already by 10 days for vessels induced by low VEGF, but not until 17 days for those induced by medium VEGF, despite similar pericyte coverage, while aberrant structures induced by high VEGF always regressed. Data represent the mean ± SEM of individual images (n) acquired from three muscles/group; 2 weeks (B): V Low saline, n = 9; V Med saline, n = 13; V High saline, n = 8; V Low TRAP,n = 28; V Med TRAP,n = 22; V High TRAP,n = 30; 3 weeks (C): V Low saline, n = 9; V Med saline, n = 11; V High saline, n = 19; V Low TRAP,n = 43; V Med TRAP,n = 14; V High TRAP,n = 19; *P < 0.05 and ***P < 0.001 (all versus Ctrl) by one-way ANOVA with Bonferroni multiple comparisons test; 2 weeks (B): V Low TRAP versus Ctrl P < 0.0001; 3 weeks (C): V Low TRAP versus Ctrl P < 0.0001; V Med TRAP versus Ctrl P = 0.0158.
Figure 1   Bovine heart mitochondria were incubated at the indicated pH in absence or presence of 200 μM DPC, treated with 1% (w/v) digitonin and F-ATP synthase was immunoprecipitated with an anti-F-ATP synthase Ab followed by 15% SDS-PAGE. β subunit and CyPD content were detected by Western blotting. The ratio between CyPD and the corresponding β subunit is reported in the lower part of the panel, referring to pH 7.4 values as 100%. Data are an average ± s.e. of 3 independent experiments.   B,C EDTA-SMP were solubilized at the indicated pH values, extracted with 1% (w/v) digitonin and subjected to BN-PAGE in order to separate dimers (Vd) and monomers (Vm), which were identified by Coomassie blue (panel B) or in-gel activity staining (panel C) and analyzed by densitometry, which is reported in the bottom part of each panel. Values report the dimer/monomer ratio, where the ratio at pH 7.4 was taken as 100%. Data are an average ± s.e. of 3 independent experiments. The p-values calculated with the Student t test are shown * p≤0.05 *** p≤0.001. D Sequence of peptides 95-113 of OSCP before (left panel) and after reaction with DPC (right panel) obtained from tryptic digests of the SDS-PAGE band corresponding to the expected molecular mass of OSCP in immunoprecipitated F-ATP synthase. Fragments of the series b and y identified in the LC-MS/MS analysis are indicated on the sequence of the peptides. H112 and H112CeT are indicated in red boldface. Ions y4-y17 show a mass shift of +72 Da in the modified peptide.
The GBS pigment toxin/lipid toxin is pro-inflammatory and cytotoxic to immortalized THP-1 monocyte-derived macrophages.Figure 3A, B WT THP-1macrophages were treated with GBS WT, ΔcylE, ΔcovR, or ΔcovRΔcylE at an MOI of 1 and incubated for 4 h. Cytotoxicity was measured by LDH release (A), and IL1β release in supernatants was measured by Luminex assay (B).Data information: Data from three independent experiments performed in triplicate are shown, error bars ± SEM. Significance was determined using Bonferroni's multiple comparison test following ANOVA. (A) n = 3, **P = 0.0080 for WT versus ΔcovR; **P = 0.0016 for ΔcovR versus ΔcovRΔcylE. (B) n = 3, *P = 0.03 for WT versus ΔcovR; *P = 0.02 for ΔcovR versus ΔcovRΔcylE. (C) n = 3, ****P < 0.0001. (D) n = 3, *P = 0.01. C, D WT THP-1macrophages were incubated with various concentrations of GBSpigment or control ΔcylE extract for 4 h. Cytotoxicity was measured by alamar blue assay (C), and IL1β release from pigment- or ΔcylE extract-treated cells was measured by Luminex assay (D).(C) n = 3, ****P < 0.0001. (D) n = 3, *P = 0.01.
a, Wild-type, apg12-1 mutant and Δapg12 cells were cultured in nitrogen-starvation medium containing 1 mM PMSF.figf1Wild-type, apg12-1 mutant and Δapg12 cells were cultured in nitrogen-starvation medium containing 1 mM PMSF. After incubation for 6 h, cells were observed under a phase-contrast microscope. Arrows indicate autophagic bodies. b, Amino-acid sequence of Apg12. c, Wild-type (squares) and Δapg12 (circles) were cultured in nitrogen-starvation medium and their viability was determined by phloxine B staining. d, Quantification of autophagic activity of wild-type and Δapg12 cells by alkaline phosphatase (ALP) assay before (black bars) and after (white bars) nitrogen starvation for 4 h. Error bars indicate s.d. of three independent experiments. e, Homology between Apg12 and potential human and C. elegans counterparts. C. elegans U32305 is 46% similar and 22% identical to amino acids 67-186 of yeast Apg12. A human cDNA (THC173313) encodes a protein that is 59% similar and 32% identical to amino acids 102-186 of Apg12.
PGE2 induces HDAC5 hyperphosphorylation and concomitant MEF2-activation via PKD.Figure 3 A,B NRVMs were infected with recombinant adenovirus encoding Flag-HDAC5 (A) or not infected (B) and serum starved for 20 hours. The cells were pretreated with the PKC-inhibitor bisindolylmaleimide I (2 μM) or the PKD-inhibitor BPKDi (3 μM) for 20 minutes and stimulated with 100 nM endothelin-1 (ET1) or 100 μM phenylephrine (PE) or 1 μM PGE2 for 4 hours, as indicated. HDAC phosphorylation was detected by immunoblotting with the anti-HDAC5 phospho-Ser-498 antibody, phosphorylation of PKD was assessed using anti-phospho-PKD antibodies (Ser-744/Ser-748 and Ser-916), representative images are shown from 3 independent experiments  C NRVMs were infected with the 3xMEF2-Luc reporter and serum-starved for 20 hours. The cells were pretreated with the PKD-inhibitor BPKDi (3 μM) or the CamKII-inhibitor AIP (1 μM) for 20 minutes and stimulated with DMSO or 1 μM PGE2 for 24 hours. N=3, * represents significant interaction between the two treatments (P<0.05, Two-Way ANOVA, BPKDi, P<0.0001; AIP, P=0.2197), values are mean±s.e.m. The exact n and P values can be also found in the Source Data excel file for Figure 3
CWH43 mutation increases ventricular volume and causes gait dysfunction in mice.Figure 3A. Scatter plot comparing ventricular volume from CWH43WT/WT (wild type, WT), heterozygous CWH43WT/M533, homozygous CWH43M533/M533 and CWH43M533/A530 mice at 6 months. Ventricular volume was calculated from T2-weighted MR images of the mouse brain using a custom automated computer algorithm. Horizontal bars indicate the mean of the measurements in each column. Statistical significance for each mutant mouse line compared to WT was determined using the unpaired t-test (P=0.0015 for CWH43WT/M533; P=0.0014 for CWH43M533/M533; P=0.006 for CWH43M533/A530). B. Representative 3D volumetric MR images of mouse brains from 6 month old CWH43WT/WT, CWH43M533/M533 and CWH43M533/A530 mice. LV = lateral ventricle, 3rd V = third ventricle, 4th V = fourth ventricle. Scale bar is approximately 1 mm. C. Quantitative gait analysis at 7 months of age revealed increased sway among homozygous CWH43M533/M533 mice, (*P=0.03, n=5) and compound heterozygous CWH43M533/A530 (*P=0.034, n=4) mice when compared to wild type CWH43WT/WT mice (n=5). Sway (the distance between the hind paws during walking) was measured repeatedly for individual mice in each group during a constrained unidirectional walk. Data shown are the mean ± SD for each group. Statistical significance was determined using the unpaired t-test. D. Box plot showing rotarod performance data for CWH43WT/WT, CWH43WT/M533, CWH43M533/M533 and CWH43M533/A530 mice at 7 months of age. Data shown are the mean, 1st quartile, 3rd quartile, minimum and maximum for each group of mice. Statistical significance was determined using the unpaired t-test. When compared to wild type CWH43WT/WT mice (n=10), balance time on the rotarod was decreased significantly among heterozygous CWH43WT/M533 mice (*P=0.04, n=8), homozygous CWH43M533/M533 mice (*P=0.03, n=7), and compound heterozygous CWH43M533/A530 mice (*P=0.03, n=4).
Figure 5Distribution of the potential RNA editing sites found in 4SP thymocytes. The top 12 categories of biological processes, which are ranked according to the p-value, for the genes that contained potential RNA editing sites at the 4SP stage. Fisher's exact test was used for the statistical analysis. GO; gene ontology. RNA editing in the 3′UTR (C) and intronic region (D) of each gene indicated in the figures with their chromosomal location was validated by direct Sanger sequencing and the frequency of editing was compared among wild-type (WT) DP, WT 4SP thymocytes, and 4SP thymocytes isolated from CD4+ cell-specific Adar1 knockout (Cd4cre A1fl/fl) mice. The relative expression of each interferon-stimulated gene (ISG) indicated in the figure in sorted DP and 4SP thymocytes isolated from WT and Cd4cre A1fl/fl mice. Values represent the relative gene expression normalized to GAPDH mRNA and are displayed as the mean ± SEM (n = 3; Mann-Whitney U-test, *p < 0.05). The mean value for the relative expression of each ISG in WT DP thymocytes was set as one. Immunoblot analysis of total and phosphorylated STAT1 expression in thymocytes isolated from WT and Cd4cre A1fl/fl mice (n = 2 for each group). The expression of GAPDH is shown as a reference.
Figure 2A Schema of the expression vectors of ENTREP and ITCH. SS, signal sequence; CD20, CD20-homology extracellular domain; TM, transmembrane domain; C2, C2 domain; WW, tryptophan-tryptophan domain; HECT, the HECT domain; C830A, the catalytically inactive mutation. Red bar, cytoplasmic domain of ENTREP and a bait for the yeast two-hybrid screening. B, C The immunoprecipitation analysis. Co-precipitation was impaired by either the WW domain deletion of ITCH or the mutation in the PPxY motifs of ENTREP. Data shown are representative of at least two independent experiments. IP, immunoprecipitation; IB, immunoblot. D The immunofluorescence staining of Cos7 cells transiently transfected with indicated vectors. Merged, merged images of FLAG, Halo and DAPI staining. Scale bar, 20mm. Data shown are representative of at least three independent experiments. E Representative images of the proximity ligation assay (PLA) using Cos7 cells transfected with indicated vectors. PLA signals, granular dots of green color, were presented with DAPI images. Arrowheads, the plasma membrane. Scale bar, 10mm. F The box-and-whisker plot presentation of the PLA. A minimum of 10 cells per condition were counted from two independent experiments. P-values obtained by Student's t-tests and P < 0.05 was considered as statistically significant. * P < 0.05. Box-whisker plot represents the interquartile range (25th and 75th percentiles) as a box and the median as a line. The maximn and minimun values within 1.5 x interquartile range are shown as whiskers.
a, COS7 cells stably expressing yellow fluorescent protein (YFP)–ATG5 were transfected with RFP–SEC61&bgr and teal fluorescent protein (TFP)-mito, and immunostained for TOMM20.f2a, COS7 cells stably expressing yellow fluorescent protein (YFP)-ATG5 were transfected with RFP-SEC61b; and teal fluorescent protein (TFP)-mito, and immunostained for TOMM20. b, Time-lapse images of COS7 cells transfected with YFP-ATG5, RFP-SEC61b; and TFP-mito under starved conditions. c, d, Distribution of distances of YFP-ATG5 dots fromthe centre to the surface of the ER (c) and to the surface of mitochondria (d). e, f, Distances of YFP-ATG5 dots over their lifetime for data from c (e) and d (f), respectively. All scale bars, 2 µm.
GATA4 overexpression triggers cardiomyocyte proliferation in vitro and improves myocardial regeneration at P7 in vivo.Figure 4(A) Representative immunoblot for GATA4 and actin from neonatal cardiomyocytes treated with control (Ad.Con) or GATA4 expressing adenovirus (Ad.GATA4). (B) Quantification of BrdU incorporation, pH3 labelling and cytokinesis in neonatal cardiomyocytes treated as shown. *p=0.0272 for BrdU incorporation, p=0.0329 for pH3 labelling, p=0.0217 for cytokinesis. (C) Immunoblot for GATA4 and GAPDH of mouse hearts treated as shown in the experimental time scale above.(D) A quantification of the immunoblot from (C) is shown; **p=0.0062. (E) Representative pictures of myocardial sections immunostained for the indicated proteins from mice 7 days after cryoinjury and treatment with Ad.Con or Ad.GATA4. The quantification of cardiomyocytes (CM) staining positive for GATA4 vs. total cardiomyocytes is shown as bar graph (E´). The number within bars indicates the number of mice analyzed in that particular group. Scale bar: 20µm. ****p<0.0001. (E) Representative pictures of myocardial sections immunostained for the indicated proteins from mice 7 days after cryoinjury and treatment with Ad.Con or Ad.GATA4. The quantification of cardiomyocytes (CM) staining positive for GATA4 vs. total cardiomyocytes is shown as bar graph (E´). The number within bars indicates the number of mice analyzed in that particular group. Scale bar: 20µm. ****p<0.0001. (F) Representative Sirius-Red stained heart sections of mice 7 days after cryoinjury (performed at P7) and treated with adenoviruses as indicated. The scar is displayed in red (arrows). Scale bar: 500 µm. The quantification of left ventricular (LV) scar area of mice 7 days after cryoinjury is shown as bar graph (F´). *p=0.0135. (G) Representative picture of a myocardial section immunostained for the indicated proteins 7 days after cryoinjury. Scale bar: 10 µm. Quantification of Ki67 positive cardiomyocytes in these mice (G´). **p=0.0023. (G) Representative picture of a myocardial section immunostained for the indicated proteins 7 days after cryoinjury. Scale bar: 10 µm. Quantification of Ki67 positive cardiomyocytes in these mice (G´). **p=0.0023. (H) Quantification of pH3 positive cardiomyocytes and cardiomyocyte (CM) cytokinesis in mice 7 days after cryoinjury; *p=0.0264, ***p=0.0006. (I) Quantification of the capillary/cardiomyocyte ratio in mice 7 days after cryoinjury. ***p=0.0002
a) The growing phagophore, first detected as an ATG5-labelled (green) sub-resolution structure on the external surface of the ER, could directly differentiate from the ER, or derive from vesicular transport and fusion events.f7(a) Three-dimensional surface rendering of deconvolved stacks of ATG5 (green) and ATG8 (magenta) domains on the forming autophagosome. Four representative objects are shown, disposed left to right according to a temporal sequence deduced from integration of real-time data on individual structures. Additional views are presented below structures I, II and IV. Note the planarity of structure I, the first morphologically informative object in the ATG5 ring formation phase, evident in two profile views. In structure II, the ATG5 torus is disposed around the aperture of the phagophore, as clearly seen with a top view of the reconstruction of the upper third of the object (arrow, green mesh for the ATG5 surface). In structure IV, corresponding to a late step in the ATG5 ring decay phase, the cavity of the neo-formed autophagosome is detectable in the additional mid-section. (b) Two-dimensional horizontal views of representative structures of type I and II, corresponding to the planes of maximum ATG5 intensity of the z-stacks. Fluorescence intensity profiles along a line crossing the confocal image confirm the disposition of ATG5-GFP and mCh-ATG8 visible in the 3D surface reconstructions. Scale bar, 2 μm.
Figure 1A. Plot of correlation coefficients generated from comparison of the genetic profiles from sre2Δ and SPCC790.03Δ (rbd2Δ) to all other profiles from the genetic interaction array. Note the high correlations among dsc1-4, sre2, and rbd2. B. Protein structural predictions for S. pombe Rbd2 (blue) and E. coli GlpG (red). Protein structures were predicted using the Phyre2 server (http://www.sbg.bio.ic.ac.uk/phyre2). Rbd2 model structure was aligned to GlpG using PyMOL. C. Protein sequence alignment of E. coli GlpG, human RHBDL4, S. pombe Rbd2, and S. cerevisiae Rbd2. Alignments were generated using Clustal Omega server (http://www.ebi.ac.uk/Tools/msa/clustalo). Predicted transmembrane domains (TM) and shared sequence motifs were used for alignment. Positions of functionally important, conserved rhomboid amino acid residues (including predicted catalytic dyad serine and histidine) are shaded in red. Residues boxed in green were identified as required for Sre1 activation from a genetic selection and were analyzed in Figures 2E and F. The starting residue numbers of the indicated sequences within their full-length proteins are given. Asterisks mark residues identical in all sequences and colons denote conservative substitutions. D. Topology model for S. pombe Rbd2 with the conserved six-pass TM. Both the N- and C-termini face the cytoplasm. The protease active site serine and histidine form a catalytic dyad between TM4 and TM6. A SHP-box, predicted to bind Cdc48, is positioned at the Rbd2 C-terminus. E. Cells carrying a plasmid expressing rbd2-6xmCherry and either of two Golgi proteins dsc2-6xGFP(dsc2-6xGFPrbd2-6xmCherry) or Sec72-6xGFP (Sec72-6xGFPrbd2-6xmCherry) were imaged by confocal fluorescence microscopy.