EMBO/sd-character-level-ner huggingface dataset:
Figure 2(A) Schematic diagram of the β-catenin domain structure and truncation mutants used in this study. The amino acid positions, truncation mutant names, and domain names are indicated. (B) Bacterially produced full-length GST-β-catenin and truncation mutant proteins were purified and reacted with Flag-Plk1 WT produced from mammalian cells in the presence of [γ-32P]ATP. Incorporation of 32P into GST-β-catenin WT and truncation mutants was confirmed by SDS-PAGE followed by autoradiography. (C) Bacterially produced β-catenin T1 WT, T3 WT, and corresponding alanine ("A") mutants were purified and reacted with Flag-Plk1 WT in the presence of [γ-32P]ATP. Incorporation of 32P onto each "A" mutant of β-catenin was confirmed by SDS-PAGE followed by autoradiography analysis. (D) Bacterially produced full-length β-catenin WT and alanine ("A") substitution mutants were purified and reacted with Flag-Plk1 WT produced from mammalian cells in the presence of [γ-32P]ATP. Incorporation of 32P into each "A" mutant or full-length WT β-catenin was confirmed by SDS-PAGE followed by autoradiography analysis. (E) The Ser60 residue of β-catenin is highly conserved among various species. Plk1 consensus phosphorylation sequence ([D/E/Q]-X-[pS/pT]-φ-X-[D/E/Q]) was shown. Ser60 is marked in red. X, any amino-acid residue; φ, hydrophobic residue. (F) A phospho-specific antibody against the β-catenin p-S60 epitope was generated and validated. Asynchronously growing HEK293T cells transfected with either Flag-β-catenin WT or S60A mutant (S60A) were harvested and immunoblotted using an anti-β-catenin p-S60 specific antibody. To confirm the specificity, either non-phospho-Ser60 peptide (+ S60 pep) or phospho-S60 peptide (+ p-S60 pep) was added to antibody solution prior to membrane incubation. (G) Asynchronously growing HEK293T cells co-transfected with Flag-β-catenin WT and either HA-Plk1 WT or control (vector only) were harvested and subjected to immunoblot analysis using the indicated antibodies. Asterisk, a nonspecific cross-reacting band. (H) HeLa CCL2 cells were treated for 18 h with 200 ng/mL nocodazole followed by treatment with either DMSO (control) or BI 2536 for an additional 1 h. The resulting cells were lysed with 2× Laemmli SDS sample buffer and used for immunoblot analysis with the indicated antibodies.
Figure 5(a-c) In situ structures of the 96-nm repeat from pig (a), horse (b), and mouse (c) sperm principal pieces. (d-f) Classification focused on the RS1 barrel revealed two distinct classes of particles, one with (top panels) and one without (middle panels) the structure. Particles with the RS1 barrel are distributed asymmetrically around the axoneme (bottom panels). (g-i) Microtubule inner proteins in axonemes from pig (g), horse (h), and mouse (i) sperm. (j-n) Microtubule inner proteins in axonemes from other cell types and organisms. Arrowheads correspond to locations of MIPs that are prominent in mammalian sperm: magenta - helical MIP in the B-tubule; pink - large A-tubule MIP; light pink - additional density associated with MIP3a.
Figure 3Electrostatic potential surface of different FGFs for representing the (HS) binding sites. HS is placed by superimposition of FGF21core, FGF9 (PDB ID: 1IHK), FGF10 (PDB ID: 1NUN), FGF19 (PDB ID: 2P23) and FGF23 (PDB ID: 2P39) onto the FGF2/FGFR1c/HS ternary complex structure (PDB ID: 1FQ9). HS is shown as a stick representation. The positive and negative charges on the protein surface are colored blue and red, respectively.
Figure 9 Figure 9. Diagram summarizes how breast cancer cells acquire their enhanced malignancy through TDLNs. TDLNs play a pivotal role in promoting breast cancer cells for distant organ metastasis. Breast cancer cells move to TDLNs where Tregs are elevated and secreted TGF-β1 to up-regulate Il-17rb expression in cancer cells (A). Expression of IL17rb in turn activated NF-κB pathway to promote malignancy for distant organ metastasis (B). Thus, the removal of TDLNs at early time point significantly reduces distant organ metastasis.
Figure 5(A) Representative images of IL-33 and p-SMAD2/3 immunostaining on WT pancreas treated with caerulein or PBS (carrier control) to induce chronic pancreatitis (scale bar: 100 μm). (B) Il33 and Smad6 mRNA levels in caerulein-treated WT pancreas compared with PBS-treated controls at the completion of chronic pancreatitis protocol (n=8 in caerulein and n=9 in PBS group for Il33, n=9 in caerulein and n=10 in PBS group for Smad6, unpaired t-test). (C) Immunoblot for p-SMAD2/3, p-SMAD1/5, SMAD6, SMAD2/3 and SMAD1 proteins in lysates from caerulein (n=3) and PBS (n=4) treated WT pancreas. GAPDH is used as the control housekeeping protein. (D) Pancreatic tumor-free survival of WT (n=4), IL-33KO (n=5) and ST2KO (n=6) mice at the completion of the DMBA/caerulein carcinogenesis protocol (log-rank test). (E) Representative H&E images of two pancreatic tissues from WT, IL-33KO and ST2KO groups treated with DMBA/caerulein (scale bar: 100 μm). (F) Representative images of IL-33 and p-SMAD2/3 immunostaining on adjacent sections of matched normal pancreas, chronic pancreatitis and PDAC collected from pancreatic cancer patients (scale bar: 100 μm). (G, H) Quantification of nuclear (G) IL-33+ and (H) p-SMAD2/3+ epithelial cells per HPF in the matched human pancreatic tissues. Each dot represents the average cell counts across three randomly selected HPF images per sample (n=18 patients, paired t-test). (I) Correlation between IL-33+ and p-SMAD2/3+ epithelial cell counts across normal pancreas, chronic pancreatitis and PDAC samples (n=54 matched samples from 18 patients, t-test for the Pearson correlation coefficient). Data information: Graphs show mean + SD.
Sequence analysis and structure modeling of SARS-CoV-2 RBD and SARS-CoV RBD and their interactions with ACE2.Figure 1.RBD sequence alignment of SARS-CoV and SARS-CoV-2, highlighting the predominant residues that contribute to the interactions with ACE2. The distinct interactions of RBD and ACE2 for the two viruses are indicated by the down-pointing orange triangles and up-pointing red triangles, respectively. RBM residues are underlined. The one-residue insertion is indicated by the red arrow. Asterisks indicate positions of fully conserved residues. Colons indicate positions of strictly conserved residues. Periods indicate positions of weakly conserved residues. Conformational comparison between the RBD-ACE2 complex structures for SARS-CoV-2 and SARS-CoV. The RBD and ACE2 structures in the SARS-CoV-2 RBD-ACE2 complex model are shown as orange and pink tubes, respectively. The RBD and ACE2 structures in the optimized SARS-CoV RBD-ACE2 complex structure are shown as blue and green tubes, respectively. The location of noticeable subtle conformational difference is indicated by an arrow. Distinct interaction patterns in the SARS-CoV-2 and SARS-CoV RBD-ACE2 interfaces. Structures of RBD and ACE2 are shown as cartoon in pink and green colors, respectively. The side chains of the residues in both protein components, representing their unique interactions, are shown as sticks. Polar interactions (salt-bridge and hydrogen bond) are shown as blue dash line. Non-polar interactions (π-stack, π-anion, and hydrophobic interactions) are shown as orange dash line.
positive cells in aged mice reduces cardiomyocyte senescence, cardiac hypertrophy and fibrosis.Figure 7Examples of individual short axis cine-MR images of 3 or 20 month old mouse hearts. Ejection fraction and LV thickness was calculated based on manual measurements of Left ventricle epicardial and endocardial borders. % change in wall thickening calculation based on wall thickness at the 4 points indicated. Measurements were made in all cine slices at end diastole and end systole. Graphs representing data obtained from MRI analysis of >7 animals per age group. Data are mean ± S.E.M. Asterisks denote a statistical significance at P<0.05 using Mann Whitney U-test. Scale bars represent 5mm. Comparison between mean number of TAF per CM and CM area in 22 month old animals. Data are mean ± S.E.M. of n=4. >100 CMs were quantified per mouse. Schematic depicting experimental design for Figures d-f: 27 month old INK-ATTAC mice were treated 4 times with AP20187 (or Vehicle), 3 days in a row, every 2 weeks (2-month-long treatment in total) and were sacrificed afterwards for analysis. Comparison between the % of p16- or eGFP-positive CMs by RNA-in situ hybridization per plane in INK-ATTAC mice (28-29 month old) treated with vehicle or AP20187. Data are mean ± SEM of n=5 per age group. 100 CMs were analysed per mouse. Mean number of TAF (left graph) and mean % of TAF-positive nuclei (right graph) in CMs. Data are mean ± S.E.M. of n=6 per age group. 100 CMs were analysed per mouse. Mean CM area μm2. Data are mean ±S.E.M. of n=6 per age group, >150 CMs analysed per mouse. % of fibrotic area evaluated by Sirius Red staining.
USP28 is highly abundant in human squamous tumours and correlates with poor prognosis.Figure 1Expression of USP28 (left) and TP63 (right) in human lung squamous cell carcinomas (SCC, n=498), adenocarcinomas (ADC, n=513) and normal non-transformed tissue (normal SCC=338, normal ADC=348). Xena UCSC software. In box plots, the centre line reflects the median, the cross represents the mean and the upper and lower box limits indicates the first and third quartile. Whiskers extend 1.5x the IQR and outliers are marked as dots. Correlation of mRNA expression of USP28 and TP63 in lung SCC (left, n=498), ADC (right, n=513) and normal, non-transformed tissue (normal SCC=338, normal ADC=348). R: Spearmans correlation coefficient; m=Slope. Xena UCSC software. IHC analysis of USP28 and ∆Np63 protein abundance in lung cancer and non-transformed human samples (n=300). The staining intensity was quantified in arbitrary units from 0 up to 3 by three independent pathologists. In box plots, the centre line reflects the median, the cross represents the mean and the upper and lower box limits indicates the first and third quartile. Whiskers extend 1.5x the IQR and outliers are marked as dots. p-values were calculated using two-tailed t-test. Kaplan-Meier estimator of NSCLC patients stratified by USP28 (left, n=1145) and TP63 (right, n=1926) expression. p-values were calculated using log-rank test. HR: hazard ratio. Kmplot software. Kaplan-Meier estimator of lung SCC patients stratified by USP28 expression (n=271). The p-value was calculated using a logrank test. HR: hazard ratio. Kmplot software.
Figure 8Male C57BL/6J mice were injected with LV-shScrambled or shSUNCR1 lentivirus specifically into the gastrocnemius at 6 weeks of age. After two weeks of recovery, mice were fed with chow diet supplemented with 0 or 1% SUC for 6 weeks. Timeline of the experimental protocol. Male C57BL/6J mice were injected with LV-shScrambled or shSUNCR1 lentivirus specifically into the gastrocnemius at 6 weeks of age. After two weeks of recovery, mice were fed with chow diet supplemented with 0 or 1% SUC for 6 weeks. SUNCR1 protein expression in gastrocnemius from mice transfected with shSUNCR1 lentivirus or LV-shScrambled (n=3). Male C57BL/6J mice were injected with LV-shScrambled or shSUNCR1 lentivirus specifically into the gastrocnemius at 6 weeks of age. After two weeks of recovery, mice were fed with chow diet supplemented with 0 or 1% SUC for 6 weeks. The running time in low speed Male C57BL/6J mice were injected with LV-shScrambled or shSUNCR1 lentivirus specifically into the gastrocnemius at 6 weeks of age. After two weeks of recovery, mice were fed with chow diet supplemented with 0 or 1% SUC for 6 weeks. four-limb handing time Male C57BL/6J mice were injected with LV-shScrambled or shSUNCR1 lentivirus specifically into the gastrocnemius at 6 weeks of age. After two weeks of recovery, mice were fed with chow diet supplemented with 0 or 1% SUC for 6 weeks. muscle grip strength of both control and gastrocnemius-specific SUNCR1 knockdown mice. Male C57BL/6J mice were injected with LV-shScrambled or shSUNCR1 lentivirus specifically into the gastrocnemius at 6 weeks of age. After two weeks of recovery, mice were fed with chow diet supplemented with 0 or 1% SUC for 6 weeks. The enzymes activity of (F) HK, (G) LDH, (H) SDH in gastrocnemius. Male C57BL/6J mice were injected with LV-shScrambled or shSUNCR1 lentivirus specifically into the gastrocnemius at 6 weeks of age. After two weeks of recovery, mice were fed with chow diet supplemented with 0 or 1% SUC for 6 weeks. Immunoblots and quantification of MyHC-I, IIb, NFAT, and PGC-1α protein in gastrocnemius (n=3).
Working model summarising the crosstalk between Wnt/β‐catenin signalling and autophagy described in this study.Figure 9
Figure 3A) A previous structure of SARS PLpro bound to a non-hydrolysable, Lys48-linked diubiquitin probe (pdb 5e6j, (Békés et al, 2016)) explained the noted preference of PLpro for longer Lys48-linked chains. While the proximal ubiquitin unit occupies the S1 site in a highly similar fashion in SARS~Ub and SARS2~Ub structures (see B, Fig 2A, EV2), the second, distal, ubiquitin unit binds to the α2 helix of PLpro, through a common binding mode involving the ubiquitin Ile44 patch. On helix α2, a central Phe70 in SARS PLpro residue is flanked by residues involved in polar contacts. B) Structure of the SARS2 PLpro~Ub complex. The S2 site consisting of α2 helix with Phe69 residue, is fully conserved in SARS2 PLpro (Fig EV1). C) Calculated enzymatic efficiencies for K48-diUb-TAMRA cleavage, followed by fluorescence polarisation with PLpro wild-type (reproduced from Appendix Fig S1A), and PLpro F69S. (Left) Using the same concentration range of SARS2 PLpro, the F69S mutant activity could not be fitted; (middle) a higher concentration recovered an activity slightly lower as compared to PLpro cleaving ubiquitin-TAMRA (45 vs. 86 M-1s-1, compare Fig 1D, Appendix Fig S1A). (Right) A ~3-fold lower efficiency for PLpro F69S cleaving ISG15-TAMRA yields values similar to cleavage of ISG15CTD-TAMRA (Appendix Fig S1A), suggesting that the S2 site contributes the difference in binding for the N-terminal Ubl-fold. Experiments for F69S were performed in technical triplicate and biological duplicate; wild-type data is reproduced from experiments shown in Fig 1D, Appendix Fig S1A. D-F) Gel based analysis showing hydrolysis time course of triubiquitin (D), proISG15CTD (E) and proISG15 (F) using wild-type PLpro (left, wild-type gels reproduced from Appendix Fig S1B-D to enable direct comparison) or PLpro F69S (right). PLpro F69S has a strong effect on triubiquitin hydrolysis (D), no marked effect on hydrolysis of proISG15CTD (E) and reduces proISG15 cleavage to the same levels as proISG15CTD (compare E, F), Experiments were performed in duplicate, see Source Data for uncropped gels.
Figure 8RPE1 were transfected with control siRNA or CSNK1D siRNA or were treated with PF670462 prior fixation. Cells were probed with rabbit polyclonal to FAM110A and mouse monoclonal to γ-Tubulin. Representative images are shown, scale bars indicate 10 μm.   Quantification of (A). Plotted is median FAM110A intensity at spindle poles positive for γ-tubulin ± SD. Each dot represents a single spindle pole. Statistical significance was determined by ANOVA (n=3) (**** p<0.0001).   Cells expressing the wild-type or mutant EGFP-FAM110A were fixed and stained for γ-tubulin and α-tubulin and analyzed by confocal microscopy (z-stack 0.16 µm). Representative images of maximal projections of the metaphase cells are shown, scale bars indicate 10 μm. Merged images show signal of DAPI, γ-tubulin and EGFP. Insets show a single Z plane at the spindle pole. Quantification of (C). Plotted is median EGFP intensity at spindle poles positive for γ-tubulin ± SD. Each dot represents a single spindle pole. Statistical significance was determined by ANOVA (n=3) (*** p<0.001). Cells expressing the wild-type FAM110A or FAM110A-S252-255E mutant were treated with DMSO or with PF670462 for 6 h and were stained and imaged as in (C). Quantification of (E). Plotted is median EGFP intensity at spindle poles positive for γ-tubulin ± SD. Each dot represents a single spindle pole. Statistical significance was determined by ANOVA (n=3) (*** p<0.001, * p<0.05). HEK293 cells transfected with indicated constructs were treated or not with PF670462 for 6 h. Pull down from cell extracts was performed using GFP trap and binding of tubulin was determined by immunoblotting (n=2).
a) HEK293T cells were transfected with an empty vector (EV) or the indicated FLAG-tagged constructs.figf2a) HEK293T cells were transfected with an empty vector (EV) or the indicated FLAG-tagged constructs. 24 h after transfection, cells were treated with either MG132 or solvent for 3 h before collection for immunoblotting as indicated. (b) HeLa cells stably expressing FLAG-HA-tagged FBXL2 were transfected with either siRNAs targeting FBXL2 (four different siRNAs) or a non-silencing siRNA (NS). Cells were lysed, and proteins were immunoblotted as indicated. NT, non-transfected. (c) During a 72-h serum starvation, NHFs were transfected with either an siRNA targeting FBXL2 (no. 1) or a non-silencing siRNA (NS). Cells were subsequently stimulated with media containing serum and collected at the indicated time points for immunoblotting. The graph shows FBXL2 mRNA levels in the different samples analysed using real-time PCR in triplicate measurements. The values represent the ratios between FBXL2 and GAPDH mRNAs. SR, serum re-addition. (d) HEK293T cells were transfected with HA-tagged p85β, FLAG-tagged FBXL2, FLAG-tagged FBXL2(ΔF-box) or an empty vector (EV) as indicated. After immunopurification with anti-FLAG resin, in vitro ubiquitylation of p85β was performed in the presence of E1, E2s and ubiquitin (Ub). Where indicated, an excess of methylated ubiquitin (MeUb) was also added. Samples were analysed by immunoblotting with the indicated antibodies. The ladder of bands with a relative molecular mass of >85,000 (lane 3) corresponds to ubiquitylated p85β. Immunoblots of whole-cell lysates (WCL) are shown at the bottom. Uncropped images of blots are shown in Supplementary Fig. S8
Figure 5786-0 cells expressing WT or enzymatically dead (ED) GDH1 were transfected with siNC or siRNF213 for 48 hours and then were treated with or without aa starvation for 12 hours. Western blotting was performed using the indicated antibodies (A). 786-0 cells expressing WT or enzymatically dead (ED) GDH1 were transfected with siNC or siRNF213 for 48 hours and then were treated with or without aa starvation for 12 hours. The intracellular level of αKG was measured using an αKG determination kit and normalized to 786-0 cells expressing WT GDH1 transfected with siNC under normal culture conditions. Unpaired t-test was used (B). Data information: The data are represented as the means ± SEM of three independent experiments (N.S.: not significant; *p < 0.05; **p < 0.01; ***p < 0.001). 786-0 cells expressing WT or enzymatically dead (ED) GDH1 were transfected with siNC or siRNF213 for 48 hours and then were treated with or without aa starvation for 12 hours. In the above cells in (A), qPCR was used to measure the mRNA of the indicated RP genes (C). 786-0 cells expressing WT or ED GDH1 were supplemented with or without 500 μM methylated αKG for 36 hours, and then cells were collected to extract total mRNA. The mRNA levels of the indicated RP genes were tested using qPCR (D) 786-0 cells expressing WT or ED GDH1 were supplemented with or without 500 μM methylated αKG for 36 hours, and then cells were collected to extract total mRNA. WB was performed using the indicated antibodies (E). (F) Protein synthesis assays using the Cayman kit and Coomassie brilliant blue (CBB) staining were performed in the indicated cells. WB was performed using the indicated antibodies (left), and the relative intensity of protein synthesis was quantified (right). Unpaired t-test was used. Data information: The data are represented as the means ± SEM of three independent experiments (N.S.: not significant; *p < 0.05; **p < 0.01; ***p < 0.001). (G) 786-0 cells expressing WT or ED GDH1 were treated with or without aa starvation for the indicated times. Cell viability was determined. Unpaired two-tailed t-test was used. Data information: The data are represented as the means ± SEM of three independent experiments (N.S.: not significant; *p < 0.05; **p < 0.01; ***p < 0.001). 786-0 or 769-P cells were supplemented with methylated αKG (500 μM), 2HG (500 μM) or αKG (500 μM) +2HG (500 μM) for 36 hours under aa starvation conditions. Cell viability was determined (H) Data information: The data are represented as the means ± SEM of three independent experiments (N.S.: not significant; *p < 0.05; **p < 0.01; ***p < 0.001). 786-0 or 769-P cells were supplemented with methylated αKG (500 μM), 2HG (500 μM) or αKG (500 μM) +2HG (500 μM) for 36 hours under aa starvation conditions. the mRNA levels of the indicated RP genes were tested using qPCR (I). (J) KIRC clinical samples were collected for αKG measurement, and we assessed overall survival (left panel) with log-rank test according to the dividing groups of high and low intracellular levels of αKG with the best separation (right panel). Unpaired t-test was used in the right panel with ***p < 0.001.
Figure 1Figure 1. Proteome-wide identification and classification of the Arabidopsis thaliana lysine acetylome. (A) Experimental overview. (B, C) Functional classification and subcellular localization of identified lysine-acetylated proteins. Lysine-acetylated proteins identified over all experiments were classified according to MapMan categoriesOver- or underrepresentation of categories was determined using a Fisher's exact test with all proteins identified at 1% FDR as background population. Blue and red arrows mark categories significantly enriched at 5% FDR (Benjamini-Hochberg) and a 1.5-fold-change cut-off. (B, C) Functional classification and subcellular localization of identified lysine-acetylated proteins. Lysine-acetylated proteins identified over all experiments were classified according to SUBA4 localization information. Over- or underrepresentation of categories was determined using a Fisher's exact test with all proteins identified at 1% FDR as background population. Blue and red arrows mark categories significantly enriched at 5% FDR (Benjamini-Hochberg) and a 1.5-fold-change cut-off. (D) Sequence logos for all lysine acetylation sites with all proteins identified as background population (sequence logos were generated using iceLogo (Maddelein et al, 2015).
Figure 1(A) Overview of hair follicle stem and progenitor cell dynamics with transcription factors known to regulate each stage. (B) Lineage tracing of the anagen matrix from β-actin-CreER:Confetti, 7 days after induction with either a high dose of tamoxifen (TM) (top) or low dose (bottom) for single cell clonal lineages. (C) Images of skin sections at various stages of the hair cycle, immunostained for Gata6 (red), CD34 or K14 (green) and DNA (blue). (D) TM induction scheme (D) for Gata6 inducible knockout (iKO) and wild type (WT) mice over time, showing the loss of Gata6 (red) from the hair follicle. Scale bars: 30 m. (D) images (D') for Gata6 inducible knockout (iKO) and wild type (WT) mice over time, showing the loss of Gata6 (red) from the hair follicle. Scale bars: 30 m.