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. 2021 Sep 29;297(5):101223. doi: 10.1016/j.jbc.2021.101223

Figure 3.

Figure 3

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.