EMBO/sd-character-level-ner huggingface dataset:
Figure 1(A) Crystal structure of the human AR LBD core dimer based on PDB 5JJM (1 monomer in grey and 1 monomer in pink). W752, located at the interface of the AR LBD dimer, is involved in hydrogen bond formation with T756 of the neighboring LBD and thereby stabilizes the interface. R752 disrupt this stabilization by steric hindrance and charge repulsion. The side chains of both W and R are shown at position 752. A close-up of the LBD-LBD interface is given. The ligand (DHT) is depicted as spheres. (B) Acceptor photobleaching FRET after transfection of Hep3B cells with labelled WT LBD or labelled W752R mutated LBD. Representative confocal images of Hep3B cells transiently expressing WT or W752R AR in the presence of 10 nM DHT are shown below the bars. (C) Representative pictures of the AGD of 13-week-old WT male (upper left), WT female (lower left), ARLmon/Y (upper right) and AR-/Y (lower right) mice. (D) Upper panel: a representative picture of the urogenital tract of a WT male and an ARLmon/Y mouse. Lower panel: a representative picture of the testis of a WT male and an ARLmon/Y mouse. Scale bar = 1 cm. (E-F) Serum levels of T (E) and LH (F) in WT males, ARLmon/Y and AR-/Y mice at the age of 13 weeks.
Figure 3(A) A boxplot showing log2 TPM values of HSPA5 gene expression compared between tumors and matched normal samples across 23 tumor types from TCGA with normal tissue data available. The boxplot indicates the median (central band) and the interquartile range (boxes) of the expression value and whiskers indicates values that outside of the middle 50% (interquartile). The barplot on the top shows the number of normal tissue samples for each corresponding tumor type. Asterisks indicate significant differences by student's t-test after Benjamini-Hochberg multiple testing correction (FDR < 0.05). (B) A heat map showing the Spearman correlation coefficient between the log2 TPM expression of three UPR branch pathway parental genes (rows) and SCNA scores across 32 tumor types (columns). Red indicates positive correlation coefficients (r > 0) and blue indicates negative correlation coefficients (r < 0). Size indicates significance level of correlation. Gene names colored in blue belong to the PERK pathway, orange to the ATF6 pathway and green to the IRE1 pathway. (C) A heatmap showing the partial correlation between the expression of UPR branch pathway genes with SCNA levels controlling for the effect of the gene copy number change using data from GISTIC2.0. Rows depict genes from the UPR branch pathways from REACTOME and columns depict the 32 tumor types. Cells are colored in red or blue if the gene showed significant correlation with SCNA level in that tumor type (partial correlation test setting copy numbers for genes as control, after Benjamini-Hochberg multiple hypothesis correction, FDR < 0.05). Color intensity corresponds to the partial correlation coefficient between gene expression and SCNA level. The left side bar indicates pathway membership of the genes. The bottom panel shows the variances of SCNA scores for each tumor type and the number of normal tissue samples available for partial correlation analysis.
Figure 7Immunohistochemical staining of p-T172-BCL9 expression in a human HCC tissue. MN: matched normal tissues, T: tumour tissues. Scale bar represents1000 μm. IHC staining and the expression scored in a tissue array of 75 pairs of human lung cancer patient tissues. Scale bar represents 1000 μm. A representative figure of p-T172-BCL9 IHC staining in HCC to indicate the expression in mitotic or in interphase cells. Scale bar represents 100 μm. Representative figures of p-T172-BCL9 IHC staining in interphase cells of breast cancer (D) or lung cancer (E) patient tissue. Scale bars represent 100 μm. Immunoprecipitation analysis of the interaction in 5 pairs of human HCC patient tissues by a p-T172-BCL9 antibody or a BCL9 antibody. CLTC relative binding with BCL9 was determined. Log-rank (Mantel-Cox) survival analysis based on high and low p-T172-BCL9 expression scored by immunohistochemical staining in two tissue arrays with (G) 75 lung cancer samples or (H) 145 breast cancer samples.
53BP1 phase separation promotes p53 induction upon DNA damage.Figure 6Cry2-mCherry-53BP1 1203-1972 W1495A optoDroplets were induced as indicated and fixed 5 minutes later. Endogenous BRCA1 and p53 were co-stained. As in (A), comparing Cry2-mCherry-53BP1 constructs with and without the BRCT domains. Quantifications of p53 assembly into 53BP1 optoDroplets from single-cell QIBC analysis of 500-600 cells per condition are shown below with mean (solid line) and standard deviation from the mean (dashed lines) indicated. Red bars indicate the part of 53BP1 that was expressed as Cry2-mCherry fusion. Western blot of p53 induction upon DNA damage (10 Gy, 2h) with and without 0.4 M Sorbitol. qPCR of p21 induction upon DNA damage (10 Gy, 2h) with and without 0.4 M sorbitol. Mean ± standard deviation is indicated. Western blot of p21 induction upon DNA damage (10 Gy, 2h) with and without 0.4 M sorbitol. QIBC cell cycle analysis based on DNA content and nuclear Cyclin A levels upon DNA damage with and without 0.4 M sorbitol. Cells were irradiated with 2 Gy in absence or presence of 0.4 M sorbitol. After 1h of recovery, sorbitol-containing medium was exchanged with fresh medium, and cells were allowed to recover for an additional 7h. Percentages in G1 and S/G2 are provided.
Figure 5(A) Tumors with CCT gene mutations have an increased mutational burden. All cancer genome sequences in TCGA were evaluated for mutation burden and specific base substitutions. Cancers with CCT gene mutations predicted to cause a negative impact on protein function were classified as deleterious and were compared to those with no mutations in any CCT gene. Wilcoxon rank sum test applied to all paired comparisons between tumors with and without CCT gene mutations yielded p<0.001. (B) The APOBEC3 mutational signature is enriched in tumors with CCT gene mutations. Single base substitution (SBS) patterns, as defined by COSMIC Mutational Signatures v2, were evaluated in CCT-mutated and CCT non-mutated cancer genome sequences. Depicted are the mutational signatures attributable to APOBEC3 activity (SBS 2 and SBS 13). Denoted p-values determined by Wilcoxon rank sum test. (C) Quantification of APOBEC3 mutational signatures among three tumor types that have previously been associated with high APOBEC3 activity. Breast, bladder, and cervical cancer samples from the TCGA database were divided into CCT-mutated and non-mutated genomes, as above. The number of mutations contributing to SBS 2 and SBS 13 in each tumor type was quantified and displayed as an average. Comparison by Wilcoxon rank sum test yielded p-values denoted. (D) The contribution of APOBEC3 mutational signatures (SBS 2 and SBS 13) and all other SBS signatures defined by COSMIC v2 was evaluated in all tumors and independently in breast, bladder, and cervical cancer genomes within the TCGA database. A comparison of mutational signature contributions between CCT-mutated and non-mutated tumors is shown. A z test of proportions was used to compare whether the percentage of 'other' signatures is significantly different between CCT-mutated and non-mutated tumors, p-values show statistical significance in breast and cervical cancers but not in bladder cancer. Data information: Box and whisker plots depict median and interquartile range (25-75%).
Fig.Figure 4Human neural cultures transduced by Auto-hLAG3 were treated by α-synuclein PFFs 4 days post LAG3 expression induction by DOX and kept in culture for 2 or 4 weeks. Both transgenic (hLAG3 D2G40-positive in (A) and 17B4-positive in (B)) and non-transduced, wild-type neurons (selected neurons in zoomed-in insets) propagated α-synuclein and developed characteristic pS129-positive (81A-positive in (A) and EP1536Y-positive in (B)) α-synuclein aggregates. Scale bars 25 µm. Trained ilastik algorithms were used to segment pixels of 81A, EP1536Y and MAP2 stainings imaged by high-content wide-field microscope, which were used to quantify the signal of 81A-positive (C) and EP1536Y-positive (D) α-synuclein aggregates expressed as % of MAP2-positive area. Almost the entire wells (182 fields per well) were imaged for every condition and replicate and each datapoint in the plot represents the entire well. Error bars indicate mean ± SD of biological replicates (duplicates). For few conditions, unicates were used, shown as one dot. One-way ANOVA followed by Tukey's multiple comparison test demonstrated that neurons that did not express LAG3 (DOX OFF) showed no difference in α-synuclein propagation when compared to LAG3-expressing neurons (DOX ON) as demonstrated by two different pS129 α-synuclein antibodies (p=0.9998 for 81A at 2 weeks and p=0.2522 at 4 weeks; p=0.9986 for EP1536Y at 2 weeks and p=0.3042 at 4 weeks).
Secretagogin is a Ca2+ sensor protein A–A2 Secretagogin co‐existed in the majority of CRH+ nerve endings in the median eminence (ME).Figure 5A-A2 Secretagogin co‐existed in the majority of CRH+ nerve endings in the median eminence (ME). Open rectangle denotes the general location of (B, B1). B, B1 Large axon terminals in the median eminence were immunopositive for secretagogin with silver‐intensified immunogold particles (open arrowheads) associated with axonal membrane and dense‐core vesicles (see Fig D for quantitative data). Solid arrowheads denote silver deposit particles proximal to the plasmalemma. C Immunoprecipitation using an anti‐secretagogin antibody in Ca2+‐free and Ca2+‐loaded conditions was subtractively used to decipher the Ca2+‐dependent protein interactions. Silver‐staining gel is shown.C1 Ontology classification of the 99 protein hits based on primary function assignments. Unbiased MALDI‐TOF proteomics was used to identify interacting proteins. The most abundant hits (Supplementary Table S3 is referred to for details on individual proteins) are proteins implicated in vesicle fusion, trafficking, transport and formation and the regulation of vesicle exocytosis. "Other" refers to a group of proteins without known function. D Single‐cell transcriptomics was used to validate the above proteome data by clustering mRNA transcripts encoding proteins that underpin vesicular release processes (red color labels highest mRNA abundance, whereas dark blue color indicates the lack of mRNA expression). E-E3 Rab3, a family of vesicular fusion and transport proteins (Schluter et al, ), was found co‐localized with secretagogin (arrowheads) in the median eminence. Neuronal soma in the PVN lacked appreciable co‐localization. Note that neither our MALDI‐TOF analysis nor our histochemical probing allowed the precise identification of individual Rab3C‐E family members.
THEM6 interacts with multiple ER membrane components involved in protein transport.Figure 3A, Venn diagram highlighting commonly modulated proteins (p-value ≤ 0.05, FC = 1.3) in THEM6 KO 22rv1 cells (2 clones) when compared to CTL. Up-regulated proteins are on top; Down-regulated proteins are into brackets. B, Enriched cellular components commonly down-regulated in THEM6 KO 22rv1 cells (2 clones) when compared to CTL. Data information: Enrichment analysis were performed using the STRING database C, Representative electron microscopy (EM) pictures of CTL and THEM6 KO 22rv1 cells taken at low (left) and high (right) magnification. Red arrows point towards abnormal ER structure. Scale bar represents 500 nm. D, Quantification of ER membrane length, plasma membrane length and ER/plasma membrane ratio using the EM pictures from C. Data information: Data are presented as mean values +/- SD. Statistical analysis: Unpaired t-test. Data reproducibility: n = 20 cells/condition. E, Immunofluorescence showing co-localisation of THEM6 and the ER marker calreticulin in 22rv1 cells. Scale bar represents 20 µm. Data information: Data reproducibility: representative image from 3 independent biological experiments. F, Prediction of transmembrane domain in the sequence of the THEM6 protein. Topology prediction was performed using the TMHMM server G, Western blot analysis of THEM6 expression in cytoplasmic (cyto.), membrane/organelle (memb.) and nuclear fractions (nucl.) of 22rv1 cells. HSC70 and SP1 were used as cytoplasmic and nuclear-enriched markers, respectively. Data information: Data reproducibility: representative image from 3 independent biological experiments. H, Enriched cellular components in THEM6-interacting proteins following THEM6 pulldown in T6 OE HEK293 cells. Data information: Enrichment analysis were performed using the STRING database I, Western blot analysis of THEM6, AMFR, SEC61b and XPO1 expression in CRPC cells following THEM6 immunoprecipitation in CRPC cells. Data information: Data reproducibility: representative image from 2 independent biological experiments. J, Western blot analysis of CALX and CALR expression in PCa cells following THEM6 silencing. Data information: Data reproducibility: representative image from 3 independent biological experiments.
Figure 1(A) Western blot analysis wild-type or PABPC4KO HeLa cells depleted of PABPC1 by siRNA-mediated knockdown. siGFP represents a negative control. (B) Phase-contrast images of wild-type or PABPC4KO HeLa cells depleted of PABPC1 by siRNA-mediated knockdown. Scale bar, 100 μm. (C) Total numbers of viable cells in (B) were quantified with acridine orange and propidium iodide staining and graphed as a percentage of wild-type HeLa cells. Error bars represent the SEM of three biological replicates.
Figure 8A. Immunohistochemical staining of tissue arrays containing 142 breast carcinoma samples (grade I: n=29, grade II: n=69, grade III: n=44) and 20 normal mammary tissues for TRIM46 and HDAC1 expression. Representative images are shown on the left panel. The positively stained cells were analyzed, and the mean intensity was scored by Image-Pro Plus software on the right panel. In the boxplot, the central lines, the box limits, and the whiskers represented medians, 25th/75th percentile, and min/max, respectively. (*P<0.05, **P<0.01, ****P < 0.0001, one-way ANOVA). B. Total proteins from 20 paired samples of breast cancer "T" versus adjacent normal breast tissues "A" were extracted for western blotting analysis with antibodies against TRIM46 or HDAC1. C. Quantification of the results in B by normalizing the protein level of TRIM46 or HDAC1 to that of GAPDH in "T" and "A" groups (n = 20). *P < 0.05, paired two-tailed Student's t-test. D. Bioinformatics analysis of the public dataset from TCGA in breast carcinoma samples (n=1093) and normal mammary tissues (n=112) for the expression of TRIM46, BRCA1, or BLM based on the indicated stratifications (****P < 0.0001, two-tailed unpaired t-test). In the boxplot, the central lines, the box limits, and the whiskers represented medians, 25th/75th percentile, and min/max, respectively. E. Kaplan-Meier survival analysis of TCGA BRCA dataset for the correlation between overall survival of breast cancer patients and TRIM46, BRCA1, or BLM expression.
Figure 4HEK293 cells were cultured for 48h after transfection with plasmids encoding scrambled siRNA (-) or siRNA specific for CDK2 (+) followed by western blot for NICD, CDK2, CDK1, CDK4 and CDK8. β-Actin served as loading control.   Quantification of the density of western blot bands in (A) performed by ImageJ software. Data are expressed as fold changes compared to control (scrambled siRNA transfected) cell lysate. All data represent the mean ± SEM from three independent experiments. Student's t-test analysis was performed, with **p≤0.01.   HEK293 cells were cultured for 48h after transfection with plasmids encoding scrambled siRNA (-) or siRNA specific for CDK1 (+) followed by western blot for NICD, CDK1, CDK2, CDK4 and CDK8. β-Actin has been used as loading control.   Quantification of the density of western blot bands in (C) performed by ImageJ software. Data are expressed as fold changes compared to control (scrambled siRNA transfected) cell lysate. All data represent the mean ± SEM from three independent experiments. Student's t-test analysis was performed, with *p≤0.05.   Analysis of the inhibitory activity of three highly selective CDK2 inhibitors against a panel of kinases - a selection of those tested is shown here. At both 1 and 10µM, Roscovitine is able to inhibit more than 95% of CDK2 activity, but is far less effective against other kinases. Purvalanol B (0.1 and 1µM) inhibits more than 94% of CDK2 activity. At both 1 and 10 µM, GSK650394A is able to inhibit more than 98% of CDK2 activity. Source: The Kinase Profiling Inhibitor Database HEK293 cells were treated with three highly selective CDK2 inhibitors (10 µM of Roscovitine, 0.1µM of Purvalanol B and 10µM of GSK650394A) for 3 hours. Endogenous levels of NICD were detected by western blot. β-Actin served as loading control.   Quantification of the density of western blot bands in (F) using ImageJ software. Data are expressed as fold changes compared to DMSO. All data represent the mean ± SEM from three independent experiments. One-way ANOVA analysis, followed by Dunnett test, was performed, with *p≤0.05, **p≤0.01, and ns= not significant.   HEK293 cells were treated with RO-3306, a specific CDK1 inhibitor, for 3 hours at 10 µM. Levels of NICD, FBXW7, phospho-Histone H3 and Histone H3 were detected by western blot. β-Actin was used as loading control.   Quantification of the density of western blot bands in (H) using ImageJ software. Data are expressed as fold changes compared to DMSO. All data represent the mean ± SEM from three independent experiments. Student's t-test analysis was performed, with *p≤0.05.
Figure 4A,B Co-Immunoprecipitation (Co-IP) with anti-HA (A) or anti-Flag (B) was performed for the analysis of the association between HA-CCDC50 and Flag-AIM2, -NLRP3, and -ASC in cotransfected HEK293 cells. Data information: L.C, light-chain; actin was used as a loading control. All the experiments were repeated at least once. C Immunoprecipitation (IP) analysis of the interaction of NLRP3 with endogenous CCDC50 and ASC in THP-1 cells activated by LPS plus ATP. IgG was used as a negative control. Data information: actin was used as a loading control. All the experiments were repeated at least once. D Representative endogenous colocalization analysis of CCDC50 and NLRP3 in PMA-induced THP-1 cells treated with LPS and ATP. Scale bar, 10 μm. E Representative confocal microscopy image of HCT116 cells overexpressing GFP-CCDC50 and stimulated with LPS as well as ATP. Scale bar, 10 μm. F Immunoblot analysis of lysates from HEK293 cells cotransfected with Flag- AIM2, -NLRP3, -ASC and HA-CCDC50 or empty control vector. The expression levels of Flag-tagged proteins and Actin were quantitated using ImageJ software. Data information: actin was used as a loading control. All the experiments were repeated at least once. G Immunoblot analysis of HEK293 cells transfected with FLAG-NLRP3 and an increasing amount of HA-CCDC50 plasmids. Data information: actin was used as a loading control. All the experiments were repeated at least once. H Immunoblot analysis of lysates from CCDC50-KO and CCDC50-WT THP-1 cells induced with LPS plus ATP and then treated with cycloheximide (CHX) for indicated time points. The expression levels of NLRP3 and Actin were quantitated using ImageJ software. Data information: actin was used as a loading control. All the experiments were repeated at least once. I HEK293 cells were co-transfected with Flag-NLRP3 and HA-CCDC50 or empty control vector for 24 h and then treated with MG132, NH4Cl, 3-MA, CQ or DMSO for another 6 h. Data information: actin was used as a loading control. All the experiments were repeated at least once. J Immunoblot analysis of NLRP3 in CCDC50-WT and CCDC50-KO THP-1 cells stimulated with LPS in combination with ATP. Data information: actin was used as a loading control. All the experiments were repeated at least once. K Western blotting analysis of ATG5 wild-type or knockout HEK293T cells co-transfected with Flag-NLRP3 and HA-CCDC50 or empty control vector for 24 h. Data information: actin was used as a loading control. All the experiments were repeated at least once. L Immunoblot analyses of NLRP3 in HEK293 cells in the presence of CCDC50 or its mutant CCDC50 with the deletion of MIUs. Data information: actin was used as a loading control. All the experiments were repeated at least once.
Figure 3Schematic model of the electron transport chain (ETC) and targets of the inhibitors used. MMP was monitored by TMRE fluorescence at every 1 h. FCCP (200 µM) and antimycin A (50 µM) were used as pretreatments for 30 min. Data were normalized by basal state of WT A549 cells (B-D) Data at 1 h (C), 5 h (D are presented as mean ± SEM; n = 4 (B-D) biological replicates, ****p < 0.0001, **p < 0.01, *p < 0.05 by paired T-test (C), or one-way ANOVA followed by Dunnett's multiple comparison test (D MMP was monitored by TMRE fluorescence at every 1 h. FCCP (200 µM) and antimycin A (50 µM) were used as pretreatments for 30 min. Data were normalized by basal state of WT A549 cells with DMSO (E-F) before cold stress. Data at 5 h (D, F) are presented as mean ± SEM; n = 5 (E-F), biological replicates, ****p < 0.0001, **p < 0.01, *p < 0.05 one-way ANOVA followed by Dunnett's multiple comparison test (D, F). LDH release was measured after cold stress for 24 h. FCCP (200 µM), antimycin A (50 µM), mitoQ and decylQ (500 nM) were used as pretreatments for 30 min. Data are presented as mean ± SEM; n = 4 (G) , biological replicates, ****p < 0.0001, ***p < 0.001, **p < 0.01, by one-way ANOVA followed by Dunnett's or Bonferroni's multiple comparison test. whose raw value were normalized by the C11-BODIPY 581/591 without cold stress of non-treatment, are presented as mean ± SEM; n = 5, biological replicates. **p < 0.01, *p < 0.05, by one-way ANOVA followed by Dunnett's multiple comparison test. LDH release was measured after cold stress for 24 h. FCCP (200 µM), antimycin A (50 µM), mitoQ and decylQ (500 nM) were used as pretreatments for 30 min. Data are presented as mean ± SEM; n = 3 (J), biological replicates, ****p < 0.0001, ***p < 0.001, **p < 0.01, by one-way ANOVA followed by Dunnett's or Bonferroni's multiple comparison test. (H, I) Lipid peroxidation was measured by C11-BODIPY 581/591 after cold stress for 5 h. Representative plots are shown in (H). Quantification data (I), whose raw value were normalized by the C11-BODIPY 581/591 without cold stress of non-treatment, are presented as mean ± SEM; n = 5, biological replicates. **p < 0.01, *p < 0.05, by one-way ANOVA followed by Dunnett's multiple comparison test.
Ultrastructural analysis of RZZS sheets and filaments (A) Negative-stain electron microscopy analysis of heat- and MPS1-induced sheets of filament of farnesylated mChRZZS.Figure 6(A) Negative-stain electron microscopy analysis of heat- and MPS1-induced sheets of filament of farnesylated mChRZZS. Scale bar (black): 200 nm. Data information: Experiments were technically replicated at least three times. (B) mChRZZmChSF forms rings and curved single filaments rather than sheets. Scale bar: 200 nm. Data information: Experiments were technically replicated at least three times. (C) The GFP of Spindly was removed with PreScission protease, and the resulting objects were imaged by negative stain EM. Scale bar: 200 nm. Data information: Experiments were technically replicated at least three times. (D) Heat-induced mChRZZGFPSF and MPS1-induced rings of mChRZZmChSF have similar diameters. As shown in panel B, heat- and MPS1-induced rings of mChRZZmChSF also have very similar dimensions. Data information: Experiments in D were carried out once.