Figure 3

CHK1 inhibition causes increased cell death in FA pathway deficient cells. (A) Western blots comparing H2AX phosphorylation and FANCD2 monoubiquitination in FANCA mutant GM6914 cells (lanes 1 to 6) versus FANCA corrected GM6914+A cells (lanes 7 to 12). Each cell line was treated for 24 hrs with Gö6976 in a dose range from 0 to 1000 nM as indicated. The L/S ratio represents the ratio of the upper monoubiquitinated (L ong) form of FANCD2 compared to the unmodified form (S hort) as measured by densitometry. Each blot was probed for vinculin to ensure equal protein loading. (B) A graphical representation of the number of chromosomal breaks per cell as measured by metaphase spreads 72 hrs after treatment with a GFP targeted control siRNA (Bars 1 and 2) or a CHK1 targeted siRNA (Bars 3 and 4). Bars 1 and 3 represent GM6914 and bars 2 and 4 represent the isogenic corrected GM6914+A cell line. (C) A graphical representation of the number of chromosomal breaks per cell as measured by metaphase spreads 48 hrs after treatment with Gö6976 (Bars 3, 4, 7 and 8) or DMSO only (Bars 1, 2, 5 and 6). Bars 1 and 3 represent FANCG mutant PD326 cells and bars 2 and 4 represent isogenic corrected cells. Bars 5 and 7 represent FANCE mutant (EUFA130) cells and bars 6 and 8 represent isogenic EUFA130+E corrected cells. Fifty metaphase spreads were counted per experiment. Each experiment was repeated three times with similar results. Representative experiments are shown.