EMBO/sd-character-level-ner huggingface dataset:
MXE expression is tightly regulated across tissues and development.Figure 3A) Heatmap showing all differentially expressed MXE clusters with at least 3 RPKM. Here, we used the Gini coefficient, which is a measure of the inequality among values of a frequency distribution (Ceriani & Verme, 2012) and has successfully been used to determine tissue-enriched gene sets (Zhang et al, 2017), to determine highly tissue-specific MXEs (maximum normalized Gini index of cluster) and MXEs with a broad tissue expression distribution (minimum Gini index). For each MXE cluster the percent-spliced-in (PSI) value of the ubiquitous MXE (minimum Gini index) is subtracted from the PSI value of the specific MXE (maximum Gini index of cluster) (delta PSI value) and scaled between -1 (broad tissue distribution) and 1 (highly tissue specific). Each column represents an MXE pair and each row represents MXE expression in a tissue, cell-type, or at a developmental time point. The bar graph summarizes counts where the specific MXE is 1.5 fold more spliced in than the ubiquitous MXE. B) Overview of differentially expressed genes for the Embryonic Development, ENCODE, and Human Protein Atlas datasets. C) Overview of differentially expressed MXEs for the Embryonic Development, ENCODE, and Human Protein Atlas datasets.
Proliferation of cay1∆ cellsSerial dilutions of the indicated strains were spotted on complete medium and grown at 30°C or 20°C.Figure 5Serial dilutions of the indicated strains were spotted on complete medium and grown at 30°C or 20°C. Otrt1∆ and Otrt1∆cay1∆ are strains with circularized chromosomes lacking telomeric sequence (Supplementary Fig S7). Representative images of wt and cay1∆ cells grown for 24 h at 30°C or 20°C. Scale bar, 20 μm. Quantification of cell length for strains grown in complete medium for 24 h at 30°C or 20°C. Each dot represents a single cell and median cell length (m) is indicated for each sample. Data are from three independent experiments and at least 100 cells were scored per sample in each experiment. Top: Examples of DAPI‐stained wt and cay1∆ cells grown in complete medium for 24 h at 30°C or 20°C. The white arrowhead indicates a chromosome bridge. Scale bar, 10 μm. Bottom: Quantification of chromosome bridges. Bars and error bars are averages and s.d. from three independent experiments and at least 200 cells were scored per sample in each experiment. Statistical significance was assayed using the unpaired, two‐tailed Student's t‐test. ***P 0.001 relative to 30°C.
Figure 6(A) Quantification of total distance that mice traveled for 10 min. (B) The ratios of mice explored between novel and familiar objects. Lines in boxes indicate mean levels and whiskers represent the data from minimal to maximal range. The box extends from 25% to 75% of values. Wilcoxon signed-rank test was performed to compare data to the hypothetical value 1. N=9-12 mice per group; * p<0.05, ** p<0.01, NS - no significant difference.
DAPK promotes the dissociation of beclin 1 from Bcl‐XL.Figure 5(A) HEK293 cells were transfected with Flag‐tagged beclin 1 and Bcl‐XL with or without haemagglutinin‐tagged DAPK. Beclin 1 was immunoprecipitated using Flag antibodies, and the co‐immunoprecipitated proteins, as well as the total cell extracts, were blotted using the indicated antibodies. (B) HEK293 cells were co‐transfected with Flag‐tagged T119A or T119E beclin 1 mutants and Bcl‐XL. Beclin 1 was immunoprecipitated using Flag antibodies, and the co‐immunoprecipitated proteins, as well as the total cell extracts, were blotted using the indicated antibodies. (C) HEK293 cells were transfected with 5 or 10 μg T119A or T119E beclin 1 mutants together with GFP-LC3 plasmid. After 24 h, cells were counted and lysates were prepared. (a) Representative GFP-LC3 staining. (b) The percentage of cells with punctate GFP-LC3 fluorescence per total GFP-LC3‐positive cells was quantified. Data presented are the mean±s.d. from a triplicate experiment with 100 transfected cells. The asterisks denote significance level: *P=0.01; **P=0.005. (c) Western blot analysis was performed using the indicated antibodies. (D) HEK293 cells were transfected with Bcl‐XL, Flag‐tagged beclin 1 (WT) or Flag‐tagged T119A beclin 1 mutant with or without haemagglutinin‐tagged activated DAPK (ΔCaM). Beclin 1 was immunoprecipitated using Flag antibodies, and the co‐immunoprecipitated proteins, as well as the total cell extracts, were blotted using the indicated antibodies. Levels of Bcl‐XL were quantified using NIH image software, and the ratio between immunoprecipitated and expressed Bcl‐XL was calculated. (E) Transmission electron micrographs of HEK293 cells 24 h after they were transfected with Bcl‐XL, Flag‐tagged beclin 1 and ΔCaM or pcDNA3‐luciferase as a control. The images in (c) and (d) were taken at higher magnifications of the ΔCaM treatment (see the scale bars). 'AV' indicates autophagic vacuoles. CaM, calmodulin; DAPK, death‐associated protein kinase; GFP, green fluorescent protein; GST, glutathione S‐transferase; HEK, human embryonic kidney; NIH, National Institutes of Health.
HOPS acts on apoptosis induction.Figure 3A Thymocytes from Hops+/+ and Hops−/− mice were treated with dexamethasone (Dex) and the apoptotic cells were measured by flow cytometry. The flow cytometry analysis plots are shown on the left and the data represented as a histogram on the right. Data information: the experiments were performed three times in primary cells from three different mice per condition. In A data are presented as mean ±SD. * P < 0.05; ** P < 0.01; *** P < 0.001, by two tailed Student's t-test. B HOPS was pivotal for apoptosis induction. Hops+/+ and Hops−/− MEFs were subjected to etoposide (Eto) and apoptosis was analysed by flow cytometry (left) and showed as a graph (right). Data information: the experiments were performed three times in primary cells from three different mice per condition. The experiments in MEFs were performed three times. Representative panels are shown. In data are presented as mean ±SD. * P < 0.05; ** P < 0.01; *** P < 0.001, by two tailed Student's t-test. C HOPS overexpression in MEFs promotes apoptosis. Hops+/+ and Hops−/− MEFs were transiently transfected with an empty vector (pEGFP-N1) or with a Hops encoding vector (pEGFP-N1-Hops). The apoptotic cells were measured by flow cytometry (left) on the GFP positive and negative populations and showed as a graph (right). Data information: the experiments were performed three times in primary cells from three different mice per condition. The experiments in MEFs were performed three times. Representative panels are shown. In C data are presented as mean ±SD. * P < 0.05; ** P < 0.01; *** P < 0.001, by two tailed Student's t-test. D HOPS affects p53 levels following stress in vitro. Hops+/+ and Hops−/− MEFs were treated with etoposide and harvested at the indicated time points. The p53 protein levels were semi-quantified using α-tubulin as loading control and relative p53 levels at 0 µM dosage were assumed as 1. Data information: the experiments were performed three times in primary cells from three different mice per condition. The experiments in MEFs were performed three times. Representative panels are shown.
Figure 4A. Immunoblot of extracts of RAW264.7 cells stably overexpressing flag-RKIP infected with VSV (MOI, 0.1) for the indicated times followed by immunoprecipitation (IP). WCL, whole cell lysates. B. Coimmunoprecipitation and immunoblot analysis of 293T cells transfected with vector for myc-RKIP alone or with the deletion mutants of TBK1. Numbers in parentheses indicate amino acids included in construct. FL, full length; KD, kinase domain; ULD, ubiquitin-like domain; SDD, α-helical scaffold domain; NBD, C-terminal domain.* indicates targeted band. C. Immunoblot of 293T cells transfected with vector for myc-TBK1 together with plasmid for HA-GSK3β or HA-RKIP, followed by immunoprecipitation. * indicates targeted band. D. In vitro kinase assay was carried out in the reaction mixture containing 2 mM ATP, 100 μM GST-RKIP or 100 μM GST-GFP and 5 μM flag-TBK1 at 30 °C for 30 min and then followed by immunoblotting with the indicated Antibody.
SH-BC-893 protects from ceramide-induced mitochondrial network fragmentation by disrupting intracellular trafficking.Figure 1Citrate synthase staining in MEFs treated for 3 h with BSA or palmitate (250 μM) after a 3 h pre-treatment with DMSO (control), YM201636 (YM, 800 nM), NAV2729 (NAV, 12.5 μM) or SH-BC-893 (893, 5 μM). ImageJ was used to calculate aspect ratio (B), branch length (C), and roundness (D) of mitochondria in the cells in (A) as described in Fig. EV1. PA, palmitate. MEFs were treated with BSA, palmitate (250 μM), or C16:0 CER (100 μM) for 3 h after a 1 h pre-treatment with vehicle or SH-BC-893 (5 μM) and mitochondria stained as in (A). aspect ratio (F), branch length (G), and roundness (H) of mitochondria in the cells in (E). Mean C16:0 ceramide levels in MEFs pre-treated for 3 h with vehicle (n=7), SH-BC-893 (5 μM, n=7), myriocin (myr, 10 μM, n=5), or fumonisin B1 (FB1, 30 μM, n=3) then treated with BSA or palmitate (250 μM) for 3 h. Cells treated with C16:0 CER (100 μM, n=2) for 3 h shown as a positive control. Error bars, SEM. MEFs treated as in (A) but evaluated for DRP1 (red) and citrate synthase (green) co-localization (yellow) using confocal immunofluorescence microscopy. Nuclei are labeled with DAPI (blue). Mander's overlap coefficient for DRP1 and citrate synthase (CS) for the cells in (J) calculated on a per cell basis. Representative DRP1 western blot (L) or quantification of DRP1 levels (M) using cells treated as in (J); n=3. Mean ± SD. MEFs expressing vector, TRPML-1, or GRP1-DD were treated with BSA or palmitate (250 μM) for 3 h and stained as in (A). aspect ratio (O), branch length (P), and roundness (Q) of mitochondria in the cells in (N).
Figure 5(A) A series of Flag-tagged truncated mutants of IL-2Ra substrates were expressed in HeLa cells with T7-tagged UBQLN4 and treated with (+) or without (-) 10 µM MG-132 for 4 h. Flag-IL-2Ra substrates were immunoprecipitated and their co-precipitation with UBQLN4 was analyzed. (B) T7-UBQLN4 was immunoprecipitated, and its interactions with a series of IL-2Ra proteins were detected.
Figure 3(A) Western Blot of whole cell lysate of adult mousebrain slices treated with the proteasome inhibitor MG132 (10 M) and DMSO, the GABAA receptor blocker GABAzine (GBZ, 10 M) or the voltage-gated sodium channel blocker TTX (2 M) for 4 h. Blots were stained for pMunc18S241, Munc18-1 and tubulin as loading control.(B) Quantification of (A). ERK phosphorylation of Munc18-1 in adult mousebrain slices expressed as ratio pM18S241 divided by total Munc18-1 levels. Vehicle (DMSO) was set to 1 (DMSO: 100 ± 0.4%; GBZ: 1.73 ± 0.12; TTX: 0.052 ± 0.002, n = 4, * p<0.05, ** p<0.01). (C) Western Blot of whole cell lysate of adult mousebrain slices treated with proteasome inhibitor MG132 (10 M) and MEK inhibitor PD98059 (10 M) for 4 h. Proteasome block increases the levels of phosphorylated Munc18-1 and ERK1/2, which can be prevented by MEK inhibition. Blots were stained for pMunc18S241, Munc18, pERK1/2 and ERK1/2.(D) Quantification of (C). ERK phosphorylation of Munc18-1 in adult mousebrain slices expressed as ratio pM18S241 divided by total Munc18-1 levels. Vehicle (DMSO) was set to 1 (DMSO: 100 ± 0.8%; MG132: 375 ± 14.2%; MG132 + PD98059: 24 ± 3%, n = 3, ** p<0.01). (E) DIV14 rat cortical cultures treated with type 2 mGluR agonist LY379268 (1 M) for 5 min or BDNF (100ng/ml) for 48 h in the presence of 2 M TTX and stained for pMunc18S241, Munc18, pERK and tubulin as loading control.
Bak is present in the cytosol.Figure 1A Flow chart of human tissue homogenization and subsequent fractionation into cytosolic (C) and mitochondria-containing heavy membrane (HM) fraction. With authorization by the local ethics committee, human tissue samples were obtained from the Institute of Pathology, University Hospitals of Basel. B Analysis of anti-Bax and anti-Bak antibody specificity using whole cell lysates from HCT116 wild-type, Bax KO, Bak KO and Bax/Bak DKO. After detection of Bax the membrane was redecorated with anti-Bak antibodies (Bax/Bak). Actin is used as loading control. C Western blot analysis of Bak in the heavy membrane (HM) fraction of human heart, cerebral cortex (CNS GM), kidney, brain white matter (CNS WM), lung, liver, and skeletal muscle tissue. The cytosol fraction obtained from CNS cortex (C), LDH, and Tom20 serve as fractionation and loading controls. n = 3. D Presence of Bak in the cytosol of heart, cerebral cortex (CNS GM), kidney, brain white matter (CNS WM), lung, liver, and skeletal muscle tissue analyzed by Western blot in parallel to C. The heavy membrane heart fraction (HM), LDH and Tom20 serve as fractionation and loading controls. n = 3. E Carbonate extraction of the heavy membrane protein fraction of human brain white matter (CNS WM) and lung tissue analyzed for the presence of Bak by Western blot. Membrane-associated protein supernatant (S) and membrane-integral protein pellet (P) are displayed. Smac is released from the mitochondrial inter membrane space (IMS) during carbonate extraction and CoxIV remains in the pellet. n = 2.
RNF26 modulates virus-triggered induction of type I IFNs.Figure 6(A) Effects of RNF26 knockdown on SeV-triggered activation of the IFN-β promoter. The 293 cells (2×105) were transfected with the IFN-β promoter reporter (0.1 µg) and the indicated RNAi plasmid (0.5 µg each). Thirty hours after transfection, cells were infected with SeV for 12 hours or left uninfected before reporter assays were performed. (B and C) Effects of RNF26 knockdown on virus-triggered induction of IFN-β in THP-1 cells. The THP-1-RNF26-RNAi or control cells (1×106) were infected with SeV or HSV-1 for the indicated time points or left uninfected followed by quantitative real-time PCR (B) (B and C) Effects of RNF26 knockdown on virus-triggered induction of IFN-β in THP-1 cells. The THP-1-RNF26-RNAi or control cells (1×106) were infected with SeV or HSV-1 for the indicated time points or left uninfected followed by quantitative real-time PCR (B) or ELISA analysis (C (D) Effects of RNF26 knockdown on virus-triggered induction of IFNB1 gene in THP-1 cells. The THP-1-RNF26-RNAi or control cells (1×106) were infected with VSV, EMCV or ECTV for the indicated time points or left uninfected before quantitative real-time PCR analysis was performed as in (B). (E and F) Effects of RNF26 knockdown on virus-triggered induction of TNFα in THP-1 cells. The THP-1-RNF26-RNAi or control cells (1×106) were infected with SeV or HSV-1 for the indicated time points or left uninfected followed by quantitative real-time PCR (E) and ELISA analysis (F). (G) Effects of RNF26 knockdown on virus-triggered phosphorylation of TBK1, IRF3 and IκBα. The THP-1-RNF26-RNAi or control cells (1×106) were infected with SeV or HSV-1 for the indicated time points or left uninfected, whole cell lysates were analyzed by immunoblots with anti-p-TBK1, anti-TBK1, anti-p-IRF3, anti-IRF3, anti-p-IκBα, anti-IκBα, anti-RNF26 or anti-β-actin as indicated. All experiments were repeated for at least three times with similar results. The bar graphs show mean ± S.D. (n = 3) of a representative experiment performed in triplicate.
Figure 5 (A) Scheme of the genetic strategy to delete YAP and TAZ and activate the expression of oncogenic KRas and TP53 deletion in tumor initiating cell (B) Kinetic of tumor appearance in Lgr5CREER/KRasG12D/TP53KO and Lgr5CREER/KRasG12D/TP53 YAP TAZ triple KO mice (n= 6 animals analysed per genotype)  (C-D) Immunostaining for YFP and Tunel assay (C) and immunohistochemistry for YAP and TAZ (D) in K14CREER/YAP TAZ DKO/Rosa-YFP mice and K14CREER/Rosa-YFP control mice  (E-F) Immunostaining of YFP and Tunel assay (E) and immunohistochemistry of YAP and TAZ (F) in Lgr5CREER/KRasG12D/P53 KO/Rosa-YFP mice and Lgr5CREER/KRasG12D/TP53/YAP TAZ triple KO/Rosa-YFP mice 3.5days after tamoxifen administration. Scale bars: 20 μm
Fig.Figure 5A: Cal27 and JHU022 cell lysates were immunoprecipitated with control or ILF3 antibodies and RNA was isolated from the precipitates. Relative mRNA expression of EGFR was analyzed by qRT-PCR. n = 3 biological replicates and 3 technical replicates. Data are represented as the mean ± SD, B: Cal27 and JHU029 cells were transfected with control or ILF3 siRNA and after 48hr cell lysates were subjected to Western blot analysis for EGFR and ILF3 using specific antibodies. The membrane was reprobed with an antibody against Actin as internal control. n = 3 biological replicates. C: Cal27 and JHU029 cell lysates were transfected with either siELDR or siILF3 and relative mRNA expression of Cyclin E1 was analyzed by qRT-PCR. 18S was used as internal control. n = 3 biological replicates. Data are represented as the mean ± SD, D: Control or ELDR depleted Cal27 and JHU029 cell lysates using two different siRNAs to ELDR were subjected to Western blot analysis for Cyclin E1 using specific antibody. The membrane was reprobed with an antibody against Actin as an internal control. Bottom panels show quantitation. n = 4 biological replicates. Data are represented as the mean ± SD, E: Control or ELDR overexpressed NOK lysates were subjected to Western blot analysis for Cyclin E1 using specific antibody. The membrane was reprobed with an antibody against Actin as internal control. Bottom panel shows quantitation. n = 3 biological replicates. Data are represented as the mean ± SD, F: Control or ILF3depleted Cal27 and JHU029 cell lysates were subjected to Western blot analysis for Cyclin E1 using specific antibodies. The membrane was reprobed with an antibody against Actin as internal control. Bottom panels show quantitation. n = 3 biological replicates. Data are represented as the mean ± SD, G: Cal27 and JHU029 cells were transfected with either siELDR or siILF3 and after 48hr cell lysates were subjected to Western blot analysis for phospho-RB and total RB using specific antibodies. The membrane was reprobed with an antibody against Actin as internal control. Bottom panels show quantitation. n = 3 biological replicates. Data are represented as the mean ± SD H: Control or ELDR knockdown synchronized Cal27 and JHU029 cells were harvested, fixed and stained with propidium iodide. DNA content was analyzed by flow cytometry. Results are represented cell population in G1, S, and G2/M phases of the cell cycle. Right panels show percentage of cell population in different phases of cell cycle. n = 4 biological replicates. Data are represented as the mean ± SD, I: Schematic representation of ELDR-ILF3 mediated regulation of Cyclin E1 signaling. The arrow indicates activation.
to NMDAR promoters and inhibits the expression of NMDARs.Figure 5(A) Overexpression of AAV-hTau increased binding of STAT1 to the promoter regions of GluN1, GluN2A and GluN2B gene in hippocampal CA3 extracts measured by chromatin immunoprecipitation assay (ChIP) (n=3 from three independent experiments). Data information: Data were presented as mean ± SD (two-way analysis of variance (ANOVA) followed by Bonferroni's post hoc test *, p<0.05, **, p<0.01, ***, p<0.001 vs Ctrl; ###, p<0.001 vs wildtype reporters. (B) Overexpression of hTau or wildtype STAT1 (WT-STAT1) inhibits the transcription activity of NMDARs compared with the empty vector control (Ctrl) measured by luciferase activity assay in HEK293 cells (n=3 from three independent experiments). Data information: Data were presented as mean ± SD (two-way analysis of variance (ANOVA) followed by Bonferroni's post hoc test *, p<0.05, **, p<0.01, ***, p<0.001 vs Ctrl; ###, p<0.001 vs wildtype reporters. Diagrams show the predicted GAS promoter element (GASs) for STAT1 in the promoter (-2000-+299bp) of GluN1 (C) The GASs or the mutant (MUT) plasmids were co-transfected respectively with WT-STAT1 or its empty vector (Ctrl) into HEK293 cells for 24 h, and then the luciferase activity was measured (right panels). N=4 for each group. Data information: Data were presented as mean ± SD (two-way analysis of variance (ANOVA) followed by Bonferroni's post hoc test *, p<0.05, **, p<0.01, ***, p<0.001 vs Ctrl; ###, p<0.001 vs wildtype reporters. Diagrams show the predicted GAS promoter element (GASs) for STAT1 in the promoter (-2000-+299bp) of GluN2B (E) The GASs or the mutant (MUT) plasmids were co-transfected respectively with WT-STAT1 or its empty vector (Ctrl) into HEK293 cells for 24 h, and then the luciferase activity was measured (right panels). N=4 for each group. Data information: Data were presented as mean ± SD (two-way analysis of variance (ANOVA) followed by Bonferroni's post hoc test *, p<0.05, **, p<0.01, ***, p<0.001 vs Ctrl; ###, p<0.001 vs wildtype reporters. Diagrams show the predicted GAS promoter element (GASs) for STAT1 in the promoter (-2000-+299bp) of GluN2A (G, I). The GASs or the mutant (MUT) plasmids were co-transfected respectively with WT-STAT1 or its empty vector (Ctrl) into HEK293 cells for 24 h, and then the luciferase activity was measured (right panels). N=4 for each group. Data information: Data were presented as mean ± SD (two-way analysis of variance (ANOVA) followed by Bonferroni's post hoc test *, p<0.05, **, p<0.01, ***, p<0.001 vs Ctrl; ###, p<0.001 vs wildtype reporters.
a) Genomic structure of ROCK1 (exon 4–6 only) with targeted allele, Flp-deleted allele and Cre-deleted allele.f6(a) Genomic structure of ROCK1 (exon 4-6 only) with targeted allele, Flp-deleted allele and Cre-deleted allele. (b) PCR assay using tail genomic DNA from three different genotypes, separated on 0.7% agarose gel (top). Western blot analysis of ROCK1 expression in the heart tissue from three different genotypes using anti-ROCK1 antibody (bottom). β-Actin was used for protein loading control in each lane. (c) Mice were fed or starved for 16 h, and heart samples were prepared in lysis buffer. Resulting proteins were resolved by SDS-PAGE and blotted against ROCK1, LC3, autophagy marker p62 and β-actin. (d) ROCK1-deficient hearts or control hearts from animals fed or starved for 16 h were subjected to electron microscopy to identify autophagosome structures. Arrows indicate autophagic vacuoles. Scale bar, 500 nm. (e) Hearts from fed and starved ROCK1 WT and KO mice were sectioned, fixed in acetone and immunofluorescence assay was performed against endogenous LC3. Data are representative of two independent experiments from each genotype. Scale bar, 50 μm.
Figure 5(A-C) Immunoblot analysis (A), immunofluorescence images (B) and quantification of the percentage of cells with one CP110 dot (C, n = 3 independent experiments) for RPE1 cells transfected with the indicated siRNAs, cultured in serum-free medium for 48 h, and stained with antibodies against CP110 and acetylated α-tubulin and DAPI. To quantify the percentage of cells with one CP110 dot (C), >180 cells were analyzed for each experiment. Scale bar, 1 µm. (D) Immunoblot analysis of CP110 and β-actin in RPE1 cells transfected with control or CP110 siRNAs and cultured in serum-free medium for 48 h. (E, F) Immunofluorescence images (E) and quantification of the percentage of ciliated cells (F, n = 3 independent experiments) for RPE1 cells transfected with the indicated siRNAs, cultured in a serum-starved condition, and stained with antibodies against γ-tubulin and acetylated α-tubulin and DAPI. To quantify the percentage of ciliated cells (F), >120 cells were analyzed for each experiment. Scale bar, 10 µm. (G, H) Immunoprecipitation and immunoblotting (G) and quantification (H, n = 3 mice) showing the CP110-CEP97 interaction in the retinal tissues of wild-type and Enkd1 knockout mice. The intensity of each CEP97 band was normalized to that of the β-actin band. (I, J) Immunoprecipitation and immunoblotting (I) and quantification (J, n = 3 independent experiments) showing the CP110-CEP97 interaction in serum-starved RPE1 cells transfected with control or ENKD1 siRNAs. The intensity of each CEP97 band was normalized to that of the β-actin band.