Analysis of the mechanism of cell death and the cell cycle effect of CHK1 inhibition in FA pathway deficient cells. (A) An annexin V/PI staining cell death assay to assess mode of cell death in HeLa cells treated with siRNA targeting GFP, FANCA, and Gö6976. Twenty-four hrs after siRNA transfection, the cells were treated with 500 nM Gö6976 or DMSO for an additional 48 hrs. Cells were then collected and subjected to annexin V/PI staining. Cells were also treated with cisplatin 10 μM for 24 hrs as a positive apoptotic control. (B) FA pathway knockdown activates CHK1 and accumulation of cells in the G2 phase of the cell cycle. Western blots measuring phosphorylation of CDC25C, CHK1, and monoubiquitination of FANCD2 in HeLa cells treated with control siRNA (lanes 1 and 2), siRNA targeting FANCA (lanes 3 and 4), and siRNA targeting BRCA2 (lanes 5 and 6). Cells were either treated for 24 hrs with Gö6976 (lanes 2, 4, 6) or DMSO control (lanes 1, 3, 5). Membranes were probed for vinculin to ensure equal loading. (C) Cell cycle analysis of HeLa cells treated with control GFP targeted siRNA, FANCA targeted siRNA or BRCA2 targeted siRNA followed by Gö6976 or DMSO control for 24 hrs. The mitotic population was assessed by measuring histone H3 phosphorylation by flow cytometry. Populations of cells at G1 (2N), S (between 2N and 4N), G2/M (4N), G2 (4N with no histone H3 staining), and M (4N with phosphohistone H3 staining) are given as a percentage of total cell population. Values are calculated from duplicate experiments.