FA cells are hypersensitive to CHK1 silencing and inhibition. (A) Response of isogenic FANCA deficient (GM6914) and proficient (GM6914+A) lines to CHK1 silencing. Cells were treated with siRNA targeting GFP (bars 1 and 2), CHK1-1 siRNA (bars 3 and 4) or CHK1-2 siRNA (bars 5 and 6). Western blot analysis: GM6914 (lanes 1, 3); GM6914+A (lanes 2, 4). (B) Response of FANCA (GM6914), FANCD2 (PD20 cells), and FANCG (PD326) deficient lines to CHK1 inhibition by Gö6976. Cells were exposed to Gö6976 for 24 hrs and then incubated for 5 days before viability was determined by the Cell Titer-Glo assay (Promega). (C) A FA pathway deficient ovarian cancer cell line is hyper-sensitive to CHK1 inhibition. A 14 day colony count assay comparing the response of FANCF deficient 2008 ovarian cancer cells (bars 1, 3, 5, 7, 9, 11, 13, 15, and 17) versus FANCF corrected 2008F cells (bars 2, 4, 6, 8, 10, 12, 14, 16, and 18) after treatment with Gö6976, UCN-01, and siRNA against CHK1, PRKCα, PRKCβ1, and GFP. Viability of each target siRNA treated cell line was calculated as a percentage of the GFP siRNA treated cells. (D) Disruption of the FA pathway sensitizes cells to Gö6976. HeLa cells were transfected with the various siRNAs for 24 hrs followed by treatment by either Gö6976 (500 nM) or DMSO. Each bar represents the ratio of cellular viability between Gö6976 and DMSO treatment.