mutant bap1 constructs  (Addgene inc)

Journal: The Journal of Biological Chemistry

Article Title: BAP1 and YY1 regulate expression of death receptors in malignant pleural mesothelioma

doi: 10.1016/j.jbc.2021.101223

Figure Lengend Snippet: Expression levels of DR4 and DR5 are inversely correlated with BAP1 expression in malignant pleural mesothelioma. A , representative images of immunohistochemistry of DR4 and DR5 in a core from an MPM tissue microarray (TMA) with or without nuclear BAP1 expression (from 88 cores of 32 patients). B , semiquantitative analysis of DR4 and DR5 expression in MPM TMA cores with (n = 42) and without (n = 46) nuclear BAP1 expression. Each dot represents an average score per patient (n = 32). t Test; p = 0.024 (DR4) and p = 0.0092 (DR5). See section for details. C , immunoblots of DR4, DR5, and BAP1 protein expression in BAP1-mutant (n = 7) versus wt-BAP1 (n = 7) MPM cell lines. Duplet bands of DR5 represent two isoforms, DR5-short (DR5-S) and DR5-long (DR5-L). Sensitivity to rTRAIL treatment is indicated as font color: green sensitive (S); orange partially sensitive (PS); and red resistant (R). D , quantitative analysis of immunoblot intensity of DR4 and DR5 in wt BAP1 and BAP1-mutant MPM cell lines (DR4 t test, p = 0.046; DR5 t test, p = 0.009). Dot color indicates the sensitivity to rTRAIL treatment as shown in ( C ). E , quantitative analysis of immunoblot intensity of DR4 and DR5 in early passage MPM cells with (+) and without (−) nuclear BAP1 expression. (DR4 t test, p = 0.033; DR5 t test, p = 0.049). F , flow cytometry analysis of DR4 and DR5 cell surface expression in early passage MPM cells with (BAP1+) and without (BAP1−) nuclear BAP1 expression alongside an isotype control. BAP1, BRCA1-associated protein 1; DR, death receptor; MPM, malignant pleural mesothelioma; rTRAIL, recombinant tumor necrosis factor–related apoptosis-inducing ligand.

Article Snippet: Vectors expressing BAP1-mutant constructs were generated by site-directed mutagenesis (E0554; New England Biolabs) of the pCCL-CMV-BAP1 vector as previously described ( ).

Techniques: Expressing, Immunohistochemistry, Microarray, Western Blot, Mutagenesis, Flow Cytometry, Recombinant

Journal: The Journal of Biological Chemistry

Article Title: BAP1 and YY1 regulate expression of death receptors in malignant pleural mesothelioma

doi: 10.1016/j.jbc.2021.101223

Figure Lengend Snippet: BAP1 knockdown increases death receptor expression and TRAIL sensitivity in cancer cells. A , immunoblots of proapoptotic proteins in parental, BAP1 shRNA (shBAP1—clone 1) or empty vector (EV) shRNA transduced BAP1-wt MPM cells (H2818) across multiple time points (0, 6, 12, 24, and 48 h) post rTRAIL treatment (100 ng/ml). Duplet bands of DR5 represent two isoforms, DR5-short (DR5-S) and DR5-long (DR5-L). The bands were quantified and normalized to an average of parental cells data. B , cell viability assay of parental, shBAP1-transduced, or EV-transduced H2818 cells following treatment with a dose range of rTRAIL (0–1000 ng/ml) for 72 h. C , immunoblot analysis in BAP1-wt-clear cell renal cell carcinoma (CCRCC) cells (BB65 and Caki-1) and MPM cells (H2818) transduced with BAP1 (shBAP1 1 or shBAP1 2) or EV shRNA. The bands were quantified and normalized to EV. D , cell viability assay of EV-transduced or shBAP1-transduced wt-BAP1 CCRCC cells following treatment with a dose range of rTRAIL (0–1000 ng/ml) for 72 h. E , relative expression of DR4 and DR5 mRNA in CCRCC cells transduced with EV or shBAP1 assessed by quantitative PCR. Relative mRNA expression was normalized to beta-2-microgloblin (B2M) expression. Data shown are the mean ± SD of two experiments performed in triplicates. t test; ∗ p < 0.05, ∗∗ p < 0.01. BAP1, BRCA1-associated protein 1; MPM, malignant pleural mesothelioma; rTRAIL, recombinant tumor necrosis factor–related apoptosis-inducing ligand; TRAIL, tumor necrosis factor–related apoptosis-inducing ligand.

Article Snippet: Vectors expressing BAP1-mutant constructs were generated by site-directed mutagenesis (E0554; New England Biolabs) of the pCCL-CMV-BAP1 vector as previously described ( ).

Techniques: Expressing, Western Blot, shRNA, Plasmid Preparation, Viability Assay, Transduction, Real-time Polymerase Chain Reaction, Recombinant

Journal: The Journal of Biological Chemistry

Article Title: BAP1 and YY1 regulate expression of death receptors in malignant pleural mesothelioma

doi: 10.1016/j.jbc.2021.101223

Figure Lengend Snippet: The deubiquitinase (DUB) function of BAP1 regulates the transcription of DR4 and DR5. A , immunoblots of proapoptotic proteins in BAP1 null early passage mesothelioma cells (Meso-8T) transduced with constructs expressing wt-BAP1 (wt-BAP1), DUB-mutant BAP1 (C91A-BAP1), or a control vector (cont-vec) untreated and after 5 h of rTRAIL treatment (50 ng/ml). B , flow cytometry analysis of cell surface expression of DR4 and DR5 in Meso-8T cells transduced with constructs expressing wt-BAP1 (wt-BAP1) or one of two DUB-mutant BAP1 vectors (C91A or A95D). One-way ANOVA; ∗∗∗ p < 0.001. C , cell viability assay of Meso-8T cells transduced with wt-BAP1 or one of two DUB-mutant BAP1 vectors (C91A or A95D) following treatment with a dose range of rTRAIL (0–1000 ng/ml) for 72 h. D , relative DR4 and DR5 mRNA expression in parental Meso-8T cells and cells transduced with wt-BAP1 or C91A-BAP1. Relative mRNA expression was normalized to beta-2-microgloblin (B2M) expression. Data are shown as the mean ± SD of two experiments performed in triplicates. ∗ p < 0.05; ∗∗ p < 0.01. E , reporter assay for promoter activities of DR4 and DR5 in parental Meso-8T cells transduced with a luciferase reporter under the control of DR4 or DR5 promoter and cells further transduced with wt-BAP1 or A95D-BAP1. Firefly luciferase/Renilla luciferase ratios were determined as relative luciferase activities. Data are shown as the mean ± SD of two experiments (n = 6 in each experiment). ∗ p < 0.05; ∗∗ p < 0.01. F , cell viability assay of wt-BAP1 H2869 cells transduced with empty vector (EV) or shBAP1 following treatment with a dose range of rTRAIL (0–1000 ng/ml) for 72 h and for shBAP1 cells further transduced with DR4 (shDR4) or DR5 shRNA (shDR5) following the same treatment. G , cell viability assay of parental BAP1 null Meso-8T early passage MPM cells transduced with wt BAP1 (wt-BAP1) or DR4 (shDR4) or DR5 shRNA (shDR5) following treatment with a dose range of rTRAIL (0–1000 ng/ml) for 72 h. +, ++; lentiviral titer. Error bars represent the SD. BAP1, BRCA1-associated protein 1; rTRAIL, recombinant tumor necrosis factor–related apoptosis-inducing ligand.

Article Snippet: Vectors expressing BAP1-mutant constructs were generated by site-directed mutagenesis (E0554; New England Biolabs) of the pCCL-CMV-BAP1 vector as previously described ( ).

Techniques: Western Blot, Transduction, Construct, Expressing, Mutagenesis, Plasmid Preparation, Flow Cytometry, Viability Assay, Reporter Assay, Luciferase, shRNA, Recombinant

Journal: The Journal of Biological Chemistry

Article Title: BAP1 and YY1 regulate expression of death receptors in malignant pleural mesothelioma

doi: 10.1016/j.jbc.2021.101223

Figure Lengend Snippet: YY1 knockdown increases the expression of DRs and rTRAIL-induced cell death. A , immunoblot analysis in BAP1-wt MPM cells (H2818, MPP89, and H2591) or CCRCC cells (BB65 and Caki-1) transduced with YY1 shRNA-clone 1 (+) or an empty vector (EV) shRNA (−). Quantitative analysis of DR4 and DR5 bands from three independent experiments was performed. Average data after normalization to tubulin were shown as bar graphs. B , relative DR4 and DR5 mRNA expression in MPM cells (H2818) and CCRCC cells (Caki-1 and BB65) transduced with YY1-shRNA or EV-shRNA. Relative mRNA expression was normalized to beta-2-microgloblin (B2M) expression. Data are shown as the mean ± SD of two experiments performed in triplicates. ∗ p < 0.05; ∗∗ p < 0.01. C , cell viability assays of BAP1-wt MPM and CCRCC cells transduced with EV shRNA or shYY1 (clone 1) following treatment with a dose range of rTRAIL (0–1000 ng/ml) or MEDI3039 (0.1–100 pM) for 72 h. Error bars represent the SD. D , immunoblot analysis in BAP1-null early passage MPM Meso-8T cells and BAP1-mutant MPM cell lines (H28 and H226) transduced with YY1 (clone 1) or EV shRNA. E , cell viability assay of EV-transduced or shYY1-transduced (clone 1) cells described in D , following treatment with a dose range of rTRAIL (0–1000 ng/ml) for 72 h. BAP1, BRCA1-associated protein 1; CCRCC, clear cell renal cell carcinoma; DR, death receptor; MPM, malignant pleural mesothelioma; rTRAIL, recombinant tumor necrosis factor–related apoptosis-inducing ligand; YY1, Ying Yang 1.

Article Snippet: Vectors expressing BAP1-mutant constructs were generated by site-directed mutagenesis (E0554; New England Biolabs) of the pCCL-CMV-BAP1 vector as previously described ( ).

Techniques: Expressing, Western Blot, Transduction, shRNA, Plasmid Preparation, Mutagenesis, Viability Assay, Recombinant

Journal: The Journal of Biological Chemistry

Article Title: BAP1 and YY1 regulate expression of death receptors in malignant pleural mesothelioma

doi: 10.1016/j.jbc.2021.101223

Figure Lengend Snippet: YY1 recruits BAP1 to the promoter regions of DR4 and DR5 and represses their transcriptional activities. A , co-immunoprecipitation (co-IP) of endogenous YY1 and BAP1 in MPM (H2818 and MPP89) and CCRCC (Caki-1) cells. B , co-IP of YY1 and BAP1 in BAP1 null early passage MPM Meso-8T cells transduced with wt BAP1 (wt-BAP1) or a control vector. C , enrichment of BAP1 and YY1 in the promoter regions of DR4 and DR5. Meso-8T cells were overexpressed with wt-BAP1, catalytically inactive BAP1-mutant (C91A-BAP1) or a control vector (cont). Chromatin immunoprecipitation (ChIP) was performed against BAP1, YY1, or IgG control followed by quantitative PCR using primers specific for promoter regions of DR4 or DR5. Error bars represent the SD. p Values are calculated to compare against IgG control using Student's t test (n = 3); ∗ p < 0.05, ∗∗ p < 0.01. D , schematic model of the transcriptional regulation of TRAIL DRs by BAP1 and YY1. BAP1, BRCA1-associated protein 1; CCRCC, clear cell renal cell carcinoma; DR, death receptor; MPM, malignant pleural mesothelioma; TRAIL, tumor necrosis factor–related apoptosis-inducing ligand; YY1, Ying Yang 1.

Article Snippet: Vectors expressing BAP1-mutant constructs were generated by site-directed mutagenesis (E0554; New England Biolabs) of the pCCL-CMV-BAP1 vector as previously described ( ).

Techniques: Immunoprecipitation, Co-Immunoprecipitation Assay, Transduction, Plasmid Preparation, Mutagenesis, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction