BRCA1 induces G2/M arrest after cisplatin injury. (A) Immunostaining of pBRCA1^S1524 (green) and DAPI (blue) in human subject–derived PTCs treated with either DMSO (Ctrl) or 25 µM cisplatin for 48 h. Quantitation of MFI for pBRCA1^S1524 staining was normalized to cell density, which was reduced by cisplatin treatment. n = 4 per condition. Scale bars = 100 µm. (B) Western blot analysis of human PTCs for pp53^(Ser15), total p53, and p21 protein levels following DMSO or 25 µM cisplatin treatment for 48 h, (molecular weight of proteins in kD) with quantitation normalized to β-actin. n = 3 per condition. (C) Western blot analysis showing BRCA1 protein expression (molecular weight of proteins in kD) in stable HKC8 cell lines expressing short hairpin control RNA (shCtrl) or either shBRCA1–1 or shBRCA1–2. (D) HKC8 cells with stable expression of shRNA (shCtrl, shBRCA1–1, and shBRCA1–2) were treated with 25 µM cisplatin for 48 h, and cell density was normalized to the untreated shCtrl cells. n = 3 per condition. (E) Flow cytometry analysis of the cell cycle phases for HKC8 cells (shCtrl, shBRCA1–1, and shBRCA1–2) treated with vehicle (Ctrl) (first panel), 25 µM cisplatin (second panel), 25 µM cisplatin and 5 µM B02, (third panel) or 5 µM B02 only (fourth panel). *P ≤ 0.05 comparing the proportion of cells in the G2/M cell cycle phase in shCtrl and shBRCA1–2 cells following cisplatin treatment. n = 3 per condition. (F) Following treatment with 25 µM of cisplatin, HKC8 cells (treated with either shCtrl, shBRCA1–1, or shBRCA1–2) were co-immunostained for BRCA1 (red), pp53^S15 (green), and nuclei with DAPI (blue). Lower panel showing zoomed-in merged images. The number of BRCA1 and pp53-positive nuclear foci were quantitated. n = 4 per condition. Scale bars = 10 µm. (G) Following treatment with 25 µM of cisplatin for 24 h, HKC8 cells (treated with shCtrl, shBRCA1–1, or shBRCA1–2) were co-immunostained for pH3 (green), γH2AX (red), and nuclei with DAPI (blue). Lower panel showing zoomed-in merged images. The ratio of pH3 and þH2AX-positive foci per cell was plotted. n = 3 per condition. Scale bars = 10 µm. (H) Following treatment with 25 µM of cisplatin for 24 h, HKC8 cells (treated with shCtrl, shBRCA1–1, or shBRCA1–2) were co-immunostained for RAD51 (green), and nuclei with DAPI (blue). The number of RAD51 foci per cell was quantitated using a threshold of four nuclear foci per cell as background. n = 3 per condition. Scale bars = 10 µm. (I) Percentage of the total number of HKC8 cells (expressing shCtrl, shBRCA1–1, or shBRCA1–2) that underwent cell death following treatment with 25 µM of cisplatin and/or 5 µM of B02. HKC8 cells stably expressing control or BRCA1 shRNA 1–1 and 1–2 were treated with 25 µM cisplatin for 24 h. n = 3 per condition. (J and K) Cell supernatants were collected, and ELISAs were performed to measure released (J) IL-6 and (K) shh ligand. n = 4 per condition. All in vitro experiments were done in triplicate and repeated two times. Unpaired t tests were performed using Welch’s correction for all statistical analyses. All data are represented as mean ± SD. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001. MFI: mean fluorescence intensity. Source data are available for this figure: SourceData F4.