EMBO/sd-character-level-ner huggingface dataset:
Figure 4(A) Extracted proteins from Huh7 cells that were untreated or treated with DFP were mixed with anti-FTMT-coupled magnetic beads and immunoblotted with the indicated antibodies (anti-NCOA4 or anti-LC3). (B) GST pulldown assay using recombinant GST-NCOA4 mixed with recombinant His-FTMT. Bound FTMT was analyzed by immunoblotting using either anti-FTMT or anti-His antibodies. (C) Immunofluorescence staining for Tom20 (red), NCOA4 (green) and FTMT (blue) in Huh7 cells before and after DFP (1 mM) treatment. Boxed areas are enlarged on the right. The white puncta indicated by white arrows show the colocalization of FTMT, NCOA4 and Tom20. Scale bar: 10 μm and 5 μm in enlarged images. (D) Super-resolution images of immunofluorescence staining for Tom20 (blue), NCOA4 (red) and FTMT (green) in Huh7 cells after DFP (1 mM) treatment, using the single-molecule fluorescence microscope. The white arrows indicate colocalization of these proteins. (E) Immunofluorescence staining for Tom20 (blue), FTMT-GFP (green), recombinant FTMTR82A-GFP (green), NCOA4-mCherry (red) and NCOA4I489A/W497A-mCherry (red) in Huh7 cells. Boxed areas are enlarged on the right. The white puncta pointed by white arrows in the overlaid picture indicate the colocalization of Tom20, FTMT-GFP and NCOA4-mCherry. The yellow puncta shown by white arrows in the overlaid picture indicate the colocalization of FTMT-GFP and NCOA4-mCherry. Scale bar: 10 μm and 5 μm in enlarged images. (F) Extracted proteins from Huh7 cells expressing the recombinant proteins as indicated were mixed with anti-GFP-coupled magnetic beads and immunoblotted with anti-mCherry and anti-GFP antibodies. Co-immunoprecipitation revealed a specific interaction of NCOA4-mCherry with FTMT-GFP or FTMTD57A-GFP, which has a substitution of alanine (A) for aspartic acid (D) at position 57 in the FTMT mitochondrial leader sequence (lanes 1 and 3). (G) Following DFP (1.0 mM) or ammonium iron sulfate (FAS) treatment as indicated, extracted proteins from Huh7 cells expressing FTMT-GFP and NCOA4-mCherry were mixed with anti-GFP-coupled magnetic beads and immunoblotted with anti-mCherry and anti-GFP antibodies.
IRF2BP1 deSUMOylation contributes to transcriptional induction of immediate early genes.Figure 3(A) Stable IRF2BP1 cell lines (knocked-down for endogenous IRF2BP1) were used to perform a microarray experiment under serum starvation and upon treatment with EGF for 1 hour. A subset of 38 EGF-dependent genes are differentially regulated in IRF2BP1 wildtype cells compared to IRF2BP1 K579R cell lines (at least 1.5-fold). Among them are DUSP1 (arrow), ATF3, Fos and Egr2. Each microarray was performed in triplicates, the bars show log2 values of the fold changes for wt cells (black bars) and for KR cells (grey bars). (B) Chromatin IP reveals association of human IRF2BP1 with the proximal DUSP1 promoter in HeLa cells. Gene architecture of human DUSP1. The primers at -243/-67 ("-67"), -473/-224 ("-224") and -1170/-961 ("-916") relative to the TSS were used for ChIP experiments. IRF2BP1 and IRF2BP2 bind to the promoter region of DUSP1 between nucleotides -243 and -67. (C) ChIP/qPCR experiments reveal preferential IRF2BP1 binding to the promoter region of DUSP1 between -243 and -67. Data show mean (bar) and individual data points from 2 biological replicates. (D) IRF2BP1 wildtype and the K579R mutant both bind the DUSP1 promoter in the absence or presence of EGF. Stable cell lines expressing IRF2BP1 wildtype or K579R were knocked-down for endogenous IRF2BP1, followed by IRF2BP1 ChIP and DUSP1 qPCR of its promoter region -243 and -67. Data show means +/- SEM from 3 biological replicates. (E) Q-PCR data after EGF treatment in IR2BP1 wildtype and K579R cell lines after knockdown of the endogenous IRF2BP1. Data show means +/- SEM from 3 biological replicates. (F) IRF2BP1 wildtype and V578A cell lines after knockdown of the endogenous IRF2BP1 were analysed for DUSP1 transcription after EGF treatment by q-PCR (left panel), data show means +/− SEM from 5 biological replicates (left panel), and IRF2BP1 protein levels. As expected, IRF2BP1 V578A is not SUMOylated (right panel). * refers to an unspecific band. (G) Q-PCR data for immediate early genes after EGF treatment in the IR2BP1 wildtype and V578A cell lines after knockdown of the endogenous IRF2BP1. Data show means +/− SEM from 4 biological replicates.
Complex I and IV enzymatic deficiencies in ∆4-CYB cells.Figure 1(A) The activities (mUnits/g of protein) of the MRC enzymes were determined by spectrophotometric kinetic measurements in WT and ∆4-CYB cells and normalized by the percentage of citrate synthase (CS) activity. Results are expressed as mean ± SD (n=4-6 biological replicates). Unpaired Student's t-test **p=0.0100; ***p=0.0002; ****p<0.0001. (B) Complex I in gel activity assays (IGA) after Blue-native gel electrophoresis (BNGE) of WT and ∆4-CYB samples solubilized either with 1.6 mg DDM/mg protein or 4 mg digitonin/mg protein. The gels were incubated in the reaction mixture for 1.5 hours (lighter signals in DDM gels) or were left to continue the reaction for 24 hours to obtain darker signals (DDM and Digitonin gels). (C) BNGE, Western blot and immunodetection, with anti-NDUFS1 (cI), anti-ATP5A (cV) and anti-SDHB (cII) antibodies, of samples from the WT cybrids and from ∆4-CYB clones E (#17.3E) and B (#17.3B).
Figure 6HeLa cells transfected with the indicated plasmids; after 24 h, cells were lysed with RIPA buffer followed by immunoprecipitation (IP) using either anti-Myc or anti-Flag agarose and Western blot with indicated antibody (n=3). HeLa and MDA-MB-231 cells were lysed with RIPA buffer, and lysates were subjected to IP using either anti-IgG, anti-BRD7 or anti-RNF146 antibodies followed by analysis with Western blot (n=3). Association of endogenous BRD7 with RNF146 in HeLa cells was performed by co-IP using anti-RNF146 antibody. Cell lysates were incubated with protein-G agarose beads conjugated with indicated antibodies, and Western blot was performed (n=3). HeLa cells with wild-type or PARP1 knockout were transfected with Myc-BRD7-WT for 24 h, their lysates were subjected to IP using anti-Myc agarose and analysed by Western blot with indicated antibodies (n=3). HeLa cells stably expressing Flag-RNF146 were transfected with either Myc-BRD7-WT or Myc-BRD7-mutant for 24 h, lysates were subjected to IP using anti-Flag agarose and analysed by Western blot with indicated antibodies (n=3).
Figure 2(A) Activated primary CD4 T cells were infected by HIV-1 (strain NLAD8) and subjected to antibody binding assay. The numbers indicate the % of Gag+ cells that are decorated by the indicated antibodies. One representative experiment is shown. (B) Antibody binding at the surface of HIV-1-infected primary CD4 T cells. Results are expressed as median fluorescence (MFI) of staining on infected (Gag+) cells. Each dot represents a single donor of CD4 T cells. n=9 donors of CD4 T cells. Data information: Error bars indicate SEM. *P<0.05, **P<0.01, Wilcoxon test. Only significant comparisons are depicted. (C) HIV-1-infected (strain NLAD8) CD4 T cells were cultured with the indicated antibody and NHS. After 24h, the surface levels of C3 and the frequency of infected cells (Gag+) were determined by flow cytometry. One representative experiment is shown. (D) C3 surface levels at the surface of HIV-1-infected primary CD4 T cells. Results are expressed as MFI of staining on infected (Gag+) cells. Each dot represents a single donor of CD4 T cells. n=7 donors of CD4 T cells. Data information: Error bars indicate SEM. *P<0.05, **P<0.01, Wilcoxon test. Only significant comparisons are depicted. (E) HIV-1-infected CD4 T cells (strain NLAD8) were cultured with the indicated antibody and normal (NHS) or heat-inactivated (HIHS) human serum. After 24h, the surface levels of C5b-9 (MAC) on infected (Gag+) cells were determined by flow cytometry. One representative experiment is shown. (F) C5b-9 (MAC) surface levels on HIV-1-infected cells (strain NLAD8). Results are expressed as MFI of staining on infected (Gag+) cells. Each dot represents a single donor of CD4 T cells. n=6 donors of CD4 T cells. Data information: Error bars indicate SEM. *P<0.05, **P<0.01, Wilcoxon test. Only significant comparisons are depicted. (G) HIV-1-infected CD4 T cells (strain NLAD8) were cultured with the indicated antibody and NHS or HIHS to determine complement-mediated cytotoxicity. After 24h, the percentage of infected (Gag+) cells in each condition was determined by flow cytometry. One representative experiment is shown. (H) Complement-mediated cytotoxicity of HIV-1-infected CD4 T cells (strain NLAD8). For each antibody, the % of CDC was calculated at 24h as the % of disappearance of Gag+ cells in NHS compared to HIHS. Each dot represents a single donor of CD4 T cells. n=7 donors of CD4 T cells. Data information: Error bars indicate SEM. *P<0.05, **P<0.01, Wilcoxon test. Only significant comparisons are depicted.
Figure 2Cells treated with 1μM Chk1i (LY2603618) for the indicated hours (h) were harvested and lysed using SDS. Indicated protein were resolved by SDS page and detected by Western blot (WB). TFIIH was used as a loading control. Percentage (%) of γ-H2AX positive cells in G1 cells after Chk1i (LY2603618) treatment for the indicated hours (h) measured using Fluorescence Activated Cell Sorting (FACS). G1 cells were considered having DAPI 2n staining and EdU negative. Percentage (%) of γ-H2AX positive cells in replicating (EdU+) cells after Chk1i (LY2603618) treatment at the indicated time points. Percentage (%) of γ-H2AX positive cells in G2/M cells after Chk1i (LY2603618) treatment. G2/M cells were considered having DAPI 4n staining and EdU negativity. Intensity of γ-H2AX measured as Relative Fluorescence Units (RFU) in early S phase cells treated with Chk1i. Mean intensity of γ-H2AX fluorescence was evaluated from EdU+ cells with 2N DAPI content. Intensity of γ-H2AX measured as Relative Fluorescence Units (RFU) in replicating cells treated with Chk1i. Mean intensity of γ-H2AX fluorescence was evaluated from EdU+. Overall, cell cycle profile of HeLa and CCNF K/O cells after Chk1i treatment. Cells with 2n DAPI staining but EdU- were gated as G1 cells; cells with >2n but <4n DAPI staining but EdU- were gated as EdU- S cells; cells with >2n but <4n DAPI staining but EdU+ were gated as EdU+ S phase; cells with 4n DAPI and pH3 S10 negative staining were gated as G2 phase and cells with 4n DAPI and pH3 S10 positive as M cells. Data from at least 3 independent replicates and plotted as Mean ± SD. Representative plots of γ-H2AX vs RPA2 signal of HeLa and CCNF K/O cells after treatment with 1μM Chk1i (LY2603618) at the indicated hours (h). Blue labeled dots represent γ-H2AX positive cells. Relates to materials section for details on representation.
Figure 4Male C57BL/6J mice were fed with chow diet supplemented with 0 and1% SUC for 6 weeks. The O2 consumption (VO2) and respiratory exchange ratio (RER) Male C57BL/6J mice were fed with chow diet supplemented with 0 and1% SUC for 6 weeks. Serum concentration of (E) NEFA in whole blood. Male C57BL/6J mice were fed with chow diet supplemented with 0 and1% SUC for 6 weeks. The enzymes activity of (F) SDH, (G) HK, and (H) LDH in gastrocnemius. Male C57BL/6J mice were fed with chow diet supplemented with 0 and1% SUC for 6 weeks. Immunoblots and quantification of of p-AMPK，PGC-1α and myoglobin in gastrocnemius. Male C57BL/6J mice were fed with chow diet supplemented with 0 and1% SUC for 6 weeks. Quantification of mitochondrial and electron transport chain (ETC) related genes expression in gastrocnemius. Male C57BL/6J mice were fed with chow diet supplemented with 0 and1% SUC for 6 weeks. OCRs were measured under basal condition in gastrocnemius.
PTPRN2 and PLCβ1 promote breast cancer metastasis.Figure 1(A,B) PTPRN2 (A) and PLCB1 (B) expression levels were determined by qRT-PCR. (C) Bioluminescence imaging quantification of lung colonization by 40,000 LM2 breast cancer cells transduced with shRNAs targeting PTPRN2 or a control hairpin. For shCntrl, sh1PTPRN2, N = 5 mice/group. For sh2PTPRN2 N = 6 mice. Right, H&amp;amp;E staining of representative lung sections. (D) Bioluminescence imaging quantification of lung colonization by 40,000 LM2 cells transfected with siRNA targeting PLCβ1 or a control siRNA. For siCntrl, N = 5 mice. For si1PLCβ1, si2PLCβ1, N = 6 mice/group. Right, H&amp;amp;E staining of representative lung sections. (E,F) PTPRN2 (E) and PLCB1 (F) levels were analyzed in human breast cancers (stages I-IV) and normal breast tissue from TissueScan qPCR Array Breast Cancer Panels II and III (Origene, N = 97). Expression levels were normalized to levels in normal tissue for each gene. (G) Kaplan-Meier curve representing overall survival of a cohort of breast cancer patients (N = 528) as a function of their primary tumor's PTPRN2 and PLCB1 expression levels (data from the TCGA Research Network (Cancer Genome Atlas, 2012)). Patients whose primary tumors' PTPRN2 and PLCB1 expression levels were higher or lower than the median of the population were classified as low (blue) or high (red) expression. (H) Kaplan-Meier curve representing distal metastasis-free survival of a cohort of breast cancer patients (N = 1609) as a function of their primary tumor's PTPRN2 and PLCB1 expression levels (Data from KMPlot (Gyorffy et al., 2010)). Patients' primary tumors' PTPRN2 and PLCB1 expression levels were classified as low (blue) or high (red) expression.
Deletion of ERp18 Regulates Cleavage of ATF6α A, B Wild type and ERp18 KO cells stably expressing ATF6α were treated with 5 µM TG for the indicated times.Figure 6A, B Wild type and ERp18 KO cells stably expressing ATF6α were treated with 5 µM TG for the indicated times. Cells were then subject to differential centrifugation to obtain membrane (A) and nuclear fractions (B) which were separated by SDS-PAGE under reducing conditions and analysed by western blotting for ATF6α. The positions of full-length ATF6α (ATF6-FL), cleaved membrane-associated ATF6α (ATF6-P) and nuclear translocated S2P cleaved ATF6α (ATF6-N) are as indicated. HDAC2 was used as a nuclear marker. (C) ATF6α was immunoisolated from wild type or ERp18 KO cells expressing ATF6α. Samples were separated by SDS-PAGE and ATF6α was detected by western blotting. HC indicates immunoglobulin heavy chains. (D, E) Blots of membrane and nuclear fractions respectively from ERp18 KO cells overexpressing ATF6α, either untreated (-) or treated (+) with 30 µM PF429242 (S1P inhibitor) prior to and following induction of ER stress with 10 mM DTT. The positions of ATF6-P, ATF6-M and ATF6-N are as indicated. The unprocessed ER-localised ATF6α (designated ER) and the O-linked glycan modified Golgi-translocated ATF6α (designated G) are indicated. HDAC2 was included as a nuclear marker. The blots in (D, E) confirm that ATF6-P is produced independently of S1P and also the absence of ATF6α processing to ATF6-N in the presence of the S1P inhibitor.
The role of Aub in GSC self-renewal is partially independent of Chk2 and requires its loading with piRNAs (A, B) Immunostaining of germaria with anti-Vasa (green) and anti-Hts (red).Figure 2(A, B) Immunostaining of germaria with anti-Vasa (green) and anti-Hts (red). DAPI (blue) was used to visualize DNA. Examples of mnkP6 and mnkP6 aubHN2/QC42 germaria are shown. White arrowheads indicate GSCs; the white arrow indicates GSC loss. (C) Quantification of the number of GSCs per non-tumorous germarium, and of germaria with GSC tumors, in the indicated genotypes. The number of scored germaria (n) is indicated. The two-tailed Student's t test and the χ2 test were used in the left and right panels, respectively. *** p-value <0.001, ** p-value <0.01, * p-value <0.05. (D-F) Immunostaining of germaria from 7 day-old females with anti-Vasa or anti-GFP (green) and anti-Hts (red). DAPI (blue) was used to visualize DNA. aubHN2/QC42; nos-Gal4/+ was used as a negative control. Examples of rescue in aubHN2/QC42; nos-Gal4/UASp-GFP-Aub germarium (E), and of lack of rescue in aubHN2/QC42; nos-Gal4/UASp-GFP-AubAA germarium (F). White arrowheads indicate GSCs; white arrows indicate GSC loss in (D) and germ cell loss in (F). Scale bar: 10 μm in A, B and D-F. (G) Quantification of mutant germaria with 0 to 1 GSC shown in (D-F). The number of scored germaria (n) is indicated. *** p-value <0.001, ns: non-significant, using the χ2 test.
Figure 3(A-C) (A) ABCA1, (B) ABCG1 and (C) LXRβ expression in islets of 8 week old mice assessed by Western blotting (mean±SEM, n=3-6 mice per genotype, relative to β-actin controls, *p<0.05, **p=<0.01, Students t-test). (D) Western blot of cell lysates of MIN6 cells after silencing of Abca12, probed with antibodies against ABCA1, ABCG1, SR-B1 and LXRβ. (E) Total cholesterol (COH) or cholesteryl esters (CE) levels in isolated pancreatic islets of mice at 8 weeks of age with deletion of Abca12 in β-cells (mean±SEM n=9, 5 and 5 mice per genotype respectively). (F) Ceramides levels in isolated pancreatic islets of mice at 8 weeks of age with deletion of Abca12 in β-cells (mean±SEM n=10, 4 and 5 mice per genotype respectively). (G) Lipid levels in MIN6 cells in which ABCA12 had been depleted by siRNA treatment (mean±SEM, n=5 (biological replicates assayed in triplicate), FC = free cholesterol, CE = cholesterol ester, Cer = ceramide, SM = sphingomyelin, TG = triacylglycerol, PC = phosphatidylcholine, *p<0.05, **p=<0.01, Students t-test). (H) Cholesterol efflux to lipid-free apoA-I (30 μg/ml, 2 h) from MIN6 cells transfected with siRNAscr or siRNAABCA12 and activated or not with TO901317 (4 μM)(mean±SEM, n=4, (biological replicates assayed in triplicate), *p=<0.05, Students t-test). (I) Cholesterol efflux to HDL (30 μg/ml, 2 h) from MIN6 cells transfected with siRNAscr or siRNAABCA12 and activated or not with TO901317 (4 μM); (mean±SEM, n=4 (biological replicates assayed in triplicate)). (J) The effect of overexpression of ABCA1 on GSIS from MIN6 cells with ABCA12 deficiency (mean±SEM, n=4 (biological replicates assayed in triplicate), *p=<0.05, Students t-test), inset shows a Western blot of cell lysates probed with anti-ABCA1 antibody in which the order of bands corresponds to the order of bars). (K) The effect of overexpression of ABCG1 on GSIS from MIN6 cells with ABCA12 deficiency (mean±SEM, n=4 (biological replicates assayed in triplicate), *p=<0.05, Students t-test, inset shows a Western blot of cell lysate probed with anti-ABCG1 anitbodies in which the order of bands corresponds to the order of bars). (L) The effect of overexpression of ABCA1 and ABCG1 on GSIS from MIN6 cells with ABCA12 deficiency (mean±SEM, n=4 (biological replicates assayed in triplicate), *p=<0.05, Students t-test). (M) The effect of activation of cells with TO901317 (4 μM) on GSIS from MIN6 cells with ABCA12 deficiency (mean±SEM, n=4 (biological replicates assayed in triplicate), *p=<0.05, Students t-test). (N) The effect of overexpression of LXRβ on GSIS from MIN6 cells with ABCA12 deficiency (mean±SEM, n=4 (biological replicates), *p=<0.05, Students t-test, inset shows a Western blot of cell lysates probed with anti-LXRβ antibody in which the order of bands corresponds to the order of bars).
Nurr1 and Foxa2 interact physically and functionally to protect mDA neurons from toxic insult.Figure 1A-C Co-localization of Nurr1 and Foxa2 proteins in mDA neurons of the adult mousemidbrain. Representative confocal sections of the ventral midbrain (A) stained with antibodies specific for TH, Nurr1, and Foxa2. The midbrains of mice at 10 weeks were cryosectioned, and stained images were taken using a confocal microscope with z-stacks through the section thickness (12 μm). Shown in (B) are individual and merged images of TH, Nurr1, and Foxa2 staining of the boxed area in (A) at higher magnification. Representative images of a single nucleus from a TH+mDA neuron of an adult midbrain section (C) co-stained with Nurr1 (red) and Foxa2 (green). Co-localization of Nurr1 and Foxa2 in the merged images was assessed by Pearson's correlation and overlap coefficient values (n = 4, right/low quadrant in C). D Immunoprecipitation (IP) assay for Nurr1 and Foxa2 protein binding. The ventral part of midbrains was dissected from 10-week-old mice, lysed, and subjected to IP. Nurr1/Foxa2 protein binding was detected by WB analysis using an anti-Foxa2 antibody in immunoprecipitates generated with anti-Nurr1 antibody (left), as well as by anti-Nurr1 WB assay in immunoprecipitates with anti-Foxa2 antibody (right). E Physical interaction between Nurr1 and Foxa2 was further assessed by a proximity ligation assay (PLA). The SN area of a midbrain section (10 weeks old) was subjected to the PLA reaction and counterstained for TH. The boxed area in the left panel exhibiting physical Nurr1/Foxa2 interaction (red) in TH+ DA neurons (green) is enlarged in the right panel. F, G Knockdown of Nurr1 and Foxa2 synergistically aggravates H2O2-induced cell death of mDA neurons in culture. Representative image for Nurr1- and Foxa2-co-expressing mDA neurons (F) used in the loss-of-function study. Shown in right panels are the individual Nurr1-, Foxa2-, and TH-stained cells of the boxed area. Nurr1- and Foxa2-co-expressing mDA neurons were formed after 9 days of differentiation in VM-NPC cultures in vitro and transduced with lentiviruses expressing shNurr1 (shN), shFoxa2 (shF), shN + shF (shNF), or shControl (shC). Three days later, the cultures were treated with H2O2 (500 μM) for 8 h and cells positive for TH (upper) and cleaved (activated) caspase-3 (lower) were counted the following day (G). Insets, high-power TH+ cell images of the boxed areas. Significantly different from the control (shC)*, shN#, shF‡ at P < 0.05, n = 5 culture wells in each group. P-values: 0.038 (shN*), 0.026 (shF*), 0.013 (shNF*), 0.036 (shNF#), and 0.042 (shNF‡) for the % TH+ cells; 0.043 (shF*), 0.037 (shNF*), 0.019 (shNF#), and 0.029 (shNF‡) for the percent cleaved caspase-3-positive cells; one-way ANOVA followed by Bonferroni post hoc test.
Fig.Figure 2A) Vacuolar membranes of wildtype (BY4741), nyv1∆ vam3∆, ypt7∆ or vma16F190Y cells were labeled with the vital dye FM4-64. A vacuole that was in a sufficiently peripheral location to be selectively photobleached was exposed to a laser pulse (white arrows indicate the bleaching area). Recovery of fluorescence in this area was assayed 10 seconds later by spinning disc confocal microscopy. Scale bar: 2 µm. B) Kinetics of FM4-64 recovery in (A). Means and s.d. are shown for 20 vacuole clusters from 3 independent experiments. C) In vivo lipid and content mixing. Vacuoles in living yeast were labeled with the indicated lumenal (CDCFDA) and membrane (FM4-64 or PX-GFP) probes. Note that CRY1 cells lack the ADE2 gene and hence naturally accumulate 5-amino-1-(5-phospho-D-ribosyl) imidazole as fluorescent fluid phase marker in the vacuolar lumen. FRAP-experiments were performed (arrows indicate the bleaching area). 1. Non-bleached lumenal area. 2. Bleached lumenal area. 3. Bleached membrane area. Scale bar: 2 µm. D and E) Kinetics of fluorescence recovery after photobleaching of the probes in (C). Numbers denote the areas shown in (C): 1. Non-bleached lumenal area. 2. Bleached lumenal area. 3. Bleached membrane area. Means and s.d. are shown for 20 vacuole clusters from 3 independent experiments.