Fig. 4

Fig. 4

BAP1 deficiency combined with SF3B1 hotspot mutation induces DNA damage response. (A) RNA‐Seq data of differentially expressed genes (DEGs) in Mel202 and OMM2.3 cells with BAP1 KO (g2) compared with vector control (vec). Data were analyzed with DESeq2 (cutoff: adjusted P‐value < 0.05). Hierarchical clustering of the DEGs was used to display the log₂‐fold changes. (B) KEGG pathways enrichment analysis for the downregulated DEGs displayed in the heatmap. Only the top 10 pathways exclusively enriched in Mel202 cells are shown. The bars represent the enrichment scores, ‐log₁₀ (adjusted P‐value). (C) Expression of DNA‐repair genes was determined by qRT‐PCR after BAP1 KO in Mel202 and OMM2.3 cells. The change of gene expression was normalized to vec control. Error bars represent SD (n = 3). (D) Immunoblots show the effect of BAP1 knockdown by siRNA on p‐ATM, total ATM, and H2AK119ub1 in Mel202 and OMM2.3. The blots shown are representative of three independent experiments. (E) Knockdown of BAP1 by siRNA led to increased 53BP1 foci and γH2AX foci in Mel202 but not OMM2.3 cells. The scale bar represents 5 μm. Graphs show mean percentage of cells with > 5 53BP1 or γH2AX foci (more than 100 cells were counted in random fields per group). The data are presented as the mean ± SD (n = 3) from one representative experiment out of three. One‐way ANOVA followed by Tukey test was used for statistical analysis. (F) Immunoblots show the effect of inducible BAP1 knockdown on p‐ATM and total ATM in OMM2.3 cells stably expressing vector, wild‐type (wt) SF3B1, or mutant SF3B1 (R625H). Stable OMM2.3 cells expressing inducible BAP1‐targeting shRNA were established. These cells were then infected with retroviruses encoding for control vector, wild‐type SF3B1, or mutant SF3B1 (R625H). Cells were treated with doxycycline (200 ng·mL⁻¹) for 72 h. The blots shown are representative of three independent experiments. (G) Effects of BAP1 KO on cell sensitivity to DNA damaging agents. Parental OMM2.3 or CRISPR‐mediated BAP1 KO clones (#1 and #2) were treated with olaparib or temozolamide with indicated concentrations for 6 days. Cell viability was determined by MTT assay. The data are presented as the mean ± SD (n = 3) from one representative experiment out of three. (H) Effects of SF3B1 mutation status on cell sensitivity to DNA damaging agents. Mel202 cells with different SF3B1 mutation status (heterozygous SF3B1 mutation, SF3B1 mut‐KO, and SF3B1 wt re‐expression in the mut‐KO cells) were treated with olaparib or temozolamide as indicated concentrations for 6 days. Cell viability was determined by MTT assay. The data are presented as the mean ± SD (n = 3) from one representative experiment out of three.