Journal: Scientific Reports

Article Title: Screening and identification of critical transcription factors involved in the protection of cardiomyocytes against hydrogen peroxide-induced damage by Yixin-shu

doi: 10.1038/s41598-017-10131-5

Figure Lengend Snippet: Transcriptional activity of Apex1 and Pbx3 and their downstream genes in H9c2 cells in response to H 2 O 2 -induced oxidative stress. The transcriptional activity of ( A ) Apex1 and ( B ) Pbx3 verified by immunofluorescence staining. Apex1 and Pbx3: green; F-actin: red, nucleus: blue. Scale bar: 50 µm. The target gene expression of ( C ) Apex1 and ( D ) Pbx3. The target genes of Apex1 and Pbx3 were all positively regulated by the corresponding TFs. Significantly different expression of a target gene is marked as P < 0.05.

Article Snippet: For the immunofluorescence staining, 10% polyformaldehyde was applied to fix the samples, followed by permeabilizing with 0.5% Triton X-100 for 30 min. After washed with PBS, the samples were treated with 10% donkey serum to block background for 60 min. Next, the primary antibody including cleaved Caspase-3 (Proteintech 25546-1-AP, 1:500), APEX1 (Proteintech 10203-1, 1:100), PBX3 (Proteintech 12571-1-AP, 1:100), was loaded at 4 °C for 24 h, followed by rhodamine phalloidin (PHDR1, cytoskeleton) and a secondary anti-body (ab 150073, 1: 200) incubation in the dark at 37 °C for 60 min.

Techniques: Activity Assay, Immunofluorescence, Staining, Targeted Gene Expression, Expressing

Journal: Scientific Reports

Article Title: Screening and identification of critical transcription factors involved in the protection of cardiomyocytes against hydrogen peroxide-induced damage by Yixin-shu

doi: 10.1038/s41598-017-10131-5

Figure Lengend Snippet: The pharmacological effects of YXS and the activity of TFs were confirmed in the hiPS-CM cell model. ( A ) YXS decreased cell apoptosis, as indicated by the decreased staining of both Annexin V (green) and PI (red). The nucleus was stained with DAPI (blue). The number of Annexin V-positive and PI-positive cells was normalized to that of the control group and expressed as a percentage of the control. ( B ) YXS reduced cleaved caspase-3, as indicated by immunofluorescence staining (green). The fluorescence intensity was normalized to that of the control group and shown in a bar graph. Cleaved caspase-3: green, F-actin: red, nucleus: blue. Scale bar: 100 μm. ( C , D ) Nuclear translocation of APEX1 and PBX3 was verified in the hiPS-CM cell model, as indicated by immunofluorescence staining of APEX1 and PBX3. APEX1 and PBX3: green; F-actin: red, nucleus: blue. The data are presented as the mean ± SD from three independent experiments. * P < 0.05 compared to the control group; # P < 0.05 compared to the model group. Scale bar: 100 μm.

Article Snippet: For the immunofluorescence staining, 10% polyformaldehyde was applied to fix the samples, followed by permeabilizing with 0.5% Triton X-100 for 30 min. After washed with PBS, the samples were treated with 10% donkey serum to block background for 60 min. Next, the primary antibody including cleaved Caspase-3 (Proteintech 25546-1-AP, 1:500), APEX1 (Proteintech 10203-1, 1:100), PBX3 (Proteintech 12571-1-AP, 1:100), was loaded at 4 °C for 24 h, followed by rhodamine phalloidin (PHDR1, cytoskeleton) and a secondary anti-body (ab 150073, 1: 200) incubation in the dark at 37 °C for 60 min.

Techniques: Activity Assay, Staining, Control, Immunofluorescence, Fluorescence, Translocation Assay

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Tandem mass tag (TMT) proteomic analysis of fetal lungs revealed differential expression of tight junction proteins in a rat model of congenital diaphragmatic hernia.

doi: 10.1016/j.biopha.2019.109621

Figure Lengend Snippet: Fig. 6. Western blotting analysis of tight junction protein expression in the lungs of fetal rats. (A, B) Representative immunoblotting and densitometric analysis of tight junction protein expression in the fetal lung. Results were normalized relative to the expres- sion of β-actin (n = 6 per group, *P < 0.05, vs. con- trol). Western blot analysis showed increased levels of Cldn3 and decreased levels of Magi1 and Myh9 in the CDH fetal lungs.

Article Snippet: The membranes were blocked in 5% nonfat dry milk for 60min and then incubated with rabbit polyclonal anti-CLDN3 (Proteintech, Chicago, USA), rabbit polyclonal anti-MAGI1 antibody (Absin, Shanghai, China), rabbit polyclonal anti-MYH9 (Proteintech Group, Chicago, USA), and mouse monoclonal β-actin (Proteintech Group, Chicago, USA) overnight at 4 °C.

Techniques: Western Blot, Expressing

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Tandem mass tag (TMT) proteomic analysis of fetal lungs revealed differential expression of tight junction proteins in a rat model of congenital diaphragmatic hernia.

doi: 10.1016/j.biopha.2019.109621

Figure Lengend Snippet: Fig. 7. IHC analysis of tight-junction proteins in the lungs of fetal rats. (A) Representative photomicrographs of IHC-staining for Cldn3, Magi1, and Myh9 in the lung sections from CDH (Left panel) and Control (right panel) fetal rats. (Original magnification ×400, scale bar = 100 μm). Cldn3 and Magi1 were mainly expressed in the epithelial cells, and some weak staining was also found in the mesenchymal cells. Myh9 protein localized to both epithelial and mesenchymal cells. (B) Semi‑quantitative analysis of IHC staining (mean density). n = 3 per group, *P < 0.05, vs. control.

Article Snippet: The membranes were blocked in 5% nonfat dry milk for 60min and then incubated with rabbit polyclonal anti-CLDN3 (Proteintech, Chicago, USA), rabbit polyclonal anti-MAGI1 antibody (Absin, Shanghai, China), rabbit polyclonal anti-MYH9 (Proteintech Group, Chicago, USA), and mouse monoclonal β-actin (Proteintech Group, Chicago, USA) overnight at 4 °C.

Techniques: Immunohistochemistry, Control, Staining

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Tandem mass tag (TMT) proteomic analysis of fetal lungs revealed differential expression of tight junction proteins in a rat model of congenital diaphragmatic hernia.

doi: 10.1016/j.biopha.2019.109621

Figure Lengend Snippet: Fig. 8. Temporal expression of tight junction mRNA in CDH lungs. (A–C) Cldn3, Magi1, and Myh9 mRNA levels in CDH lungs were determined by quantitative RT- PCR at different time points. β-actin was used as a housekeeping gene. The results were normalized relative to the same-aged control group (n = 8 per group, *P < 0.05, vs. control at the same time point).

Article Snippet: The membranes were blocked in 5% nonfat dry milk for 60min and then incubated with rabbit polyclonal anti-CLDN3 (Proteintech, Chicago, USA), rabbit polyclonal anti-MAGI1 antibody (Absin, Shanghai, China), rabbit polyclonal anti-MYH9 (Proteintech Group, Chicago, USA), and mouse monoclonal β-actin (Proteintech Group, Chicago, USA) overnight at 4 °C.

Techniques: Expressing, Quantitative RT-PCR, Control

Journal: Cancer Cell International

Article Title: Hypoxia-induced BAP1 enhances erastin-induced ferroptosis in nasopharyngeal carcinoma by stabilizing H2A

doi: 10.1186/s12935-024-03494-z

Figure Lengend Snippet: Screening for ferroptosis-related prognostic genes in HNSCC. ( A ) Twelve genes (ASNS, TRIB3, ATP5MC3, FTH1, NQO1, HSPA5, OTUB1, PRDX6, FLT3, ATG5, MAP1LC3A and BAP1) were constructed in the forest plot. ( B ) Nomogram prognostic model containing ASNS, PRDX6, FLT3, ATG5, MAP1LC3A and BAP1 in HNSCC. ( C ) Survivorship curve of the distribution of patients based on risk scores. ( D ) ROC curves at 1, 2 or 3 years. ( E ) Principal components analyses were performed on all samples. The first two principal components which explain most of the data variation are shown. ( F ) Survival status of individual HNSCC patients with the distribution of patients based on risk scores

Article Snippet: Primary antibodies include BAP1 (Proteintech, China) and H2A (CST, USA).

Techniques: Construct

Journal: Cancer Cell International

Article Title: Hypoxia-induced BAP1 enhances erastin-induced ferroptosis in nasopharyngeal carcinoma by stabilizing H2A

doi: 10.1186/s12935-024-03494-z

Figure Lengend Snippet: The prognosis risk model with six ferroptosis-related genes

Article Snippet: Primary antibodies include BAP1 (Proteintech, China) and H2A (CST, USA).

Techniques:

Journal: Cancer Cell International

Article Title: Hypoxia-induced BAP1 enhances erastin-induced ferroptosis in nasopharyngeal carcinoma by stabilizing H2A

doi: 10.1186/s12935-024-03494-z

Figure Lengend Snippet: BAP1 was upregulated in nasopharyngeal carcinoma cells under hypoxia. HNE1 and 6-10B cells were cultured under hypoxia or normoxia control for 24 h. ( A ) Six genes (BAP1, ASNS, ATG5, PRDX6, MAP1LC3A and FLT3) mRNA levels were assessed by qRT-PCR. ( B ) Protein level of BAP1 were assessed by western blot. ( C ) Luciferase reporter assay detected that HIF-1α bound to the promoter region of BAP1. ( D ) Protein level of HIF-1α and BAP1 were assessed by western blot. The data are presented as mean ± SD. * P < 0.05; *** P < 0.001

Article Snippet: Primary antibodies include BAP1 (Proteintech, China) and H2A (CST, USA).

Techniques: Cell Culture, Control, Quantitative RT-PCR, Western Blot, Luciferase, Reporter Assay

Journal: Cancer Cell International

Article Title: Hypoxia-induced BAP1 enhances erastin-induced ferroptosis in nasopharyngeal carcinoma by stabilizing H2A

doi: 10.1186/s12935-024-03494-z

Figure Lengend Snippet: Knockdown of BAP1 attenuated erastin-induced ferroptosis in nasopharyngeal carcinoma cells under hypoxia. ( A ) Western blot results confirmed BAP1 knockdown cells were constructed. ( B ) Above cells were treated with 0, 2.5, 5, 10, 20 µM erastin for 24 h under hypoxia culture environment, cell viability was assessed by CCK-8 assay. ( C - G ) HNE1 cells and 6-10B cells transfected with BAP1 knockdown or Negative control(NC) were treated with 10 µM erastin under hypoxiaor normal control culture environment. ( C ) Cell floating and death were evaluated via observation using a microscope. ( D ) ROS concentration was assessed by flow cytometer presented by MEAN FITC. ( E ) Intracellular lipid ROS was measured by using an imaging probe that becomes green fluorescent upon activation by lipid ROS. ( F ) MDA concentration was assessed by MDA assay. ( G ) GSH concentration was assessed by GSH assay. The data are presented as mean ± SD. * P < 0.05; ** P < 0.01; *** P < 0.001

Article Snippet: Primary antibodies include BAP1 (Proteintech, China) and H2A (CST, USA).

Techniques: Knockdown, Western Blot, Construct, CCK-8 Assay, Transfection, Negative Control, Control, Microscopy, Concentration Assay, Flow Cytometry, Imaging, Activation Assay, Multiple Displacement Amplification, GSH Assay

Journal: Cancer Cell International

Article Title: Hypoxia-induced BAP1 enhances erastin-induced ferroptosis in nasopharyngeal carcinoma by stabilizing H2A

doi: 10.1186/s12935-024-03494-z

Figure Lengend Snippet: Hypoxia-induced BAP1 decreased SLC7A11 expression by stabilizing H2A. ( A ) BAP1 knockdown 6-10B cells were subjected to hypoxia or normal stimulation. BAP1 and H2A expression was detected by representative immunoblotting analysis. ( B ) 6-10B cells (shBAP1 or shNC) were subjected to MG132 or solvent 3 h before harvest, BAP1 and H2A expression was detected by representative immunoblotting analysis. ( C ) 6-10B cells were subjected to hypoxia 24 h. And BAP1 antibody or normal rabbit IgG antibody were subjected to co-IP, then the indicated antibodies were used for immunoblotting analysis. ( D ) 6-10B cells transfected with BAP1 knockdown lentiviruses or empty virus were subjected to hypoxia 24 h and treated with MG132 3 h before harvest. And H2A antibody or normal rabbit IgG antibody were subjected to co-IP, then the UB antibody was used for immunoblotting analysis. ( E ) 6-10B cells transfected with BAP1 knockdown lentiviruses or empty virus were subjected to hypoxia or normal stimulation. SLC7A11 transcription level was detected by qRT-PCR. The data are presented as mean ± SD. * P < 0.05; ** P < 0.01

Article Snippet: Primary antibodies include BAP1 (Proteintech, China) and H2A (CST, USA).

Techniques: Expressing, Knockdown, Western Blot, Solvent, Co-Immunoprecipitation Assay, Transfection, Virus, Quantitative RT-PCR

Journal: Cancer Cell International

Article Title: Hypoxia-induced BAP1 enhances erastin-induced ferroptosis in nasopharyngeal carcinoma by stabilizing H2A

doi: 10.1186/s12935-024-03494-z

Figure Lengend Snippet: Knockdown of BAP1 reduced tumoricidal capacity of erastin in vivo. NPC cells(6-10B-shNC and 6-10B-shBAP1) were resuspended at 3 × 10 6 cells/mL in 200ul saline and injected into right anterior flank of male BABL/c nude mice. After 7 days, the mice were randomly divided into treatment group and control group(10 mice per group). The treatment group was treated with erastin(30 mg/kg, intraperitoneally, every day) and the control group was treated with saline(equal volume per weight, intraperitoneally, every day). ( A ) The tumor growth curves after erastin treatment in vivo. ( B ) Picture of isolated tumors. ( C ) Weight of isolated tumors. ( D ) Tumor IHC results of BAP1, H2A and SLC7A11. The data are presented as mean ± SD. * P < 0.05; ** P < 0.01; *** P < 0.001

Article Snippet: Primary antibodies include BAP1 (Proteintech, China) and H2A (CST, USA).

Techniques: Knockdown, In Vivo, Saline, Injection, Control, Isolation

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: BAP1 regulates HSF1 activity and cancer immunity in pancreatic cancer

doi: 10.1186/s13046-024-03196-4

Figure Lengend Snippet: BAP1 deficiency resulted in the resistance to immunotherapy. (A) A pie chart illustrating the copy number alterations of BAP1 in the TCGA-PAAD dataset. (B) A scatter plot depicting the correlation between BAP1 copy number alterations and mRNA levels in the TCGA-PAAD dataset. (C) Survival analysis of patients grouped based on high and low expression of BAP1 in TCGA-PAAD dataset. (D) The analysis investigated the correlation between BAP1 expression and the indicated tumor infiltrated immune cells within the PAAD tumors using the Tumor Immune Estimation Resource (TIMER, cistrome.shinyapps.io/timer). (E-F) Representative images (E) and IHC score (D) of Bap1 and Pd-l1 in KPC/KPC; Bap1 KO mice. n = 6, two-tailed unpaired Student’s t-test. (G) Western blot analysis of indicated genes of the KPC mice. (H) The comparison of pancreas mass in the specified groups. n = 6,two-tailed unpaired t test. (n.s. not significant, * P < 0.05, *** P < 0.001). (I) Survival analysis of the KPC/KPC; Bap1 KO mice in specified groups ( n = 6, ** P < 0.01). (J) FACS analysis of tumor infiltrated CD45 + CD8 + T cells, CD45 + CD4 + T cells, and CD11b + Gr1 + myeloid cells in indicated groups ( n = 6, n.s. not significant, * P < 0.05, *** P < 0.001)

Article Snippet: Immunohistochemical analysis was performed with antibodies specific for Bap1 (working dilution 1:500, Proteintech), Pd-l1 (working dilution 1:1000, Proteintech).

Techniques: Expressing, Two Tailed Test, Western Blot, Comparison

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: BAP1 regulates HSF1 activity and cancer immunity in pancreatic cancer

doi: 10.1186/s13046-024-03196-4

Figure Lengend Snippet: BAP1 represses the transcriptional activity of HSF1. (A) The volcano plot of differentially expressed genes between BAP1 deletion patients and BAP1 diploid & amplificated patients in TCGA-PAAD dataset. (B) The bar plot of the transcription factor enrich analysis of BAP1 downregulated genes. (C) The expression of BAP1 were examined via western blot in the indicated cell lines. (D-E) Cells infected with indicated sgRNAs and were screened using puromycin for 48 h, and were harvested for RT-qPCR analysis ( n = 3, n.s. not significant, *** P < 0.001) (D) and western blot (E). (F) Cells infected with indicated plasmids and were harvested for western blot. (G-H) Luciferase reporter activities of HSF1 were detected in cells with indicated conditions ( n = 5, ** P < 0.01, *** P < 0.001). (I) RT-qPCR analysis of indicated genes under conditions of BAP1 knock-out or overexpression ( n = 3, ** P < 0.01, *** P < 0.001). (J) ChIP-qPCR analysis of HSF1 on the promoter of indicated genes under conditions of BAP1 knock-out or overexpression ( n = 3, n.s. not significant, ** P < 0.01, *** P < 0.001)

Article Snippet: Immunohistochemical analysis was performed with antibodies specific for Bap1 (working dilution 1:500, Proteintech), Pd-l1 (working dilution 1:1000, Proteintech).

Techniques: Activity Assay, Expressing, Western Blot, Infection, Quantitative RT-PCR, Luciferase, Knock-Out, Over Expression, ChIP-qPCR

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: BAP1 regulates HSF1 activity and cancer immunity in pancreatic cancer

doi: 10.1186/s13046-024-03196-4

Figure Lengend Snippet: BAP1 interacts with the N-terminal of HSF1. (A-B) Endogenous Co-IP of the indicated proteins in PANC-1 and PaTu8988 cells. (C-D) Co-IP assay to detect the ubiquitination level of HSF1 in the indicated status of 293T cells. (E) Schematic depicting the domain structure and the truncation constructs. (F) 293T cells were transfected with indicated constructs and then were harvested for Co-IP and WB analysis

Article Snippet: Immunohistochemical analysis was performed with antibodies specific for Bap1 (working dilution 1:500, Proteintech), Pd-l1 (working dilution 1:1000, Proteintech).

Techniques: Co-Immunoprecipitation Assay, Ubiquitin Proteomics, Construct, Transfection

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: BAP1 regulates HSF1 activity and cancer immunity in pancreatic cancer

doi: 10.1186/s13046-024-03196-4

Figure Lengend Snippet: BAP1 facilitates HSF1 monomerization by augmenting HSP70-HSF1 interaction. (A) Western blot analysis was performed on cytosolic and nuclear fractions of PANC-1 cells infected with lentivirus expressing specified sgRNAs, followed by 48 h of puromycin selection. (B) Co-IP analysis of indicated proteins were performed on cytosolic and nuclear fractions of PANC-1 cells. (C-D) BAP1 stably knock-out PANC-1 cells were transfected with indicated constructs for 48 h and were harvested for Co-IP analysis(C) and quantification (D; n = 3 biologically independent samples) of gray value of HA-HSF1/Flag-HSF1. (E-F) Representative Co-IP analysis of the expression of indicated proteins in PANC-1 cells with transfected with indicated constructs (E) and quantification (F; n = 3 biologically independent samples) of gray value of HA-HSF1/Flag-HSF1. (G-H) PANC-1 cells were transfected with indicated constructs for 48 h and were harvested for Co-IP analysis(G) and quantification of gray value of HSP70/ HSF1. (I) Endogenous Co-IP of the indicated proteins in PANC-1 and PaTu8988 cells. (J) In the presence of proficient BAP1, BAP1 facilitates the recruitment of HSP70 by trimerized HSF1, thus promoting the dissociation of trimerized HSF1 into inactive monomeric HSF1. In the absence of BAP1, the recruitment of HSP70 by HSF1 is reduced, resulting in decreased dissociation of HSF1 and increased activity

Article Snippet: Immunohistochemical analysis was performed with antibodies specific for Bap1 (working dilution 1:500, Proteintech), Pd-l1 (working dilution 1:1000, Proteintech).

Techniques: Western Blot, Infection, Expressing, Selection, Co-Immunoprecipitation Assay, Stable Transfection, Knock-Out, Transfection, Construct, Activity Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: BAP1 regulates HSF1 activity and cancer immunity in pancreatic cancer

doi: 10.1186/s13046-024-03196-4

Figure Lengend Snippet: BAP1 inhibits the formation of SIRT1-HSF1 complex via competitive interaction. (A) PANC-1 cells were infected with lentivirus expressing specified sgRNAs and subsequently subjected to Co-IP analysis after 48 h of puromycin selection. (B-C) PANC-1 cells were transfected with indicated constructs and were harvested for Co-IP assay. (D) PANC-1 cells were infected with lentivirus expressing specified shRNAs and subsequently subjected to Co-IP analysis after 48 h of puromycin selection. (E) 293T cells were transfected with indicated constructs and were harvested for Co-IP. (F) BAP1 stably knock-out PANC-1 cells were transfected with indicated constructs for 48 h and were harvested for Co-IP analysis. (G) 293T cells were transfected with indicated constructs and were harvested for Co-IP. (H-I) Luciferase reporter activities of HSF1 were detected in PANC-1 and PaTu8988 cells under indicated conditions ( n = 5, n.s. not significant, *** P < 0.001)

Article Snippet: Immunohistochemical analysis was performed with antibodies specific for Bap1 (working dilution 1:500, Proteintech), Pd-l1 (working dilution 1:1000, Proteintech).

Techniques: Infection, Expressing, Co-Immunoprecipitation Assay, Selection, Transfection, Construct, Stable Transfection, Knock-Out, Luciferase

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: BAP1 regulates HSF1 activity and cancer immunity in pancreatic cancer

doi: 10.1186/s13046-024-03196-4

Figure Lengend Snippet: SIRT1 inhibition enhances immunotherapy efficacy in BAP1 deficient PDAC. (A) PANC-1 cells were transfected with indicated constructs for 24 h and were treated with control treatment or 200nM EX-527 for 36 h and were harvested for Co-IP. (B) BAP1 stably knock-out PANC-1 cells were harvested for the luciferase reporter assay for HSF1 after treated with indicated drugs for 36 h. (C) Diagrams depicting the process of constructing syngeneic xenograft models. (D) Line chart illustrating the progression of tumor growth in the specified groups ( n = 5, n.s. not significant, ** P < 0.01, *** P < 0.001). (E) FACS analysis of tumor infiltrated CD45 + CD8 + T cells, CD45 + CD4 + T cells, and CD11b + Gr1 + myeloid cells in indicated groups ( n = 5, n.s. not significant, ** P < 0.01, *** P < 0.001). (F) A hypothetical model illustrating BAP1 repressing HSF1 chromatin occupation via competitive interaction with K80 acetylated HSF1, countering SIRT1-mediated deacetylation and activation of HSF1

Article Snippet: Immunohistochemical analysis was performed with antibodies specific for Bap1 (working dilution 1:500, Proteintech), Pd-l1 (working dilution 1:1000, Proteintech).

Techniques: Inhibition, Transfection, Construct, Control, Co-Immunoprecipitation Assay, Stable Transfection, Knock-Out, Luciferase, Reporter Assay, Activation Assay