FIGURE 1. Depletion or inhibition of PARP1 selectively reduces the viability of BRCA1- and BRCA2-deficient ES cells.

a, Reduction of PARP1 protein levels by siRNA. A plasmid (pSUPER-eCFP-PARP1) expressing a Parp1-specific siRNA and eCFP was transfected into ES cells. A control plasmid (pSUPER-eCFP-control), expressing an unrelated scrambled siRNA and eCFP, was separately transfected. Cell lysates were analysed by western blotting with either an anti-PARP1 antibody or anti-CFP antiserum as a control. b, Reduction in viability of BRCA1- and BRCA2-deficient ES cells after Parp1-specific siRNA silencing. ES cells were transfected with either pSUPER-eCFP-PARP1 or pSUPER-eCFP-control together with a blasticidin-resistance-encoding plasmid, and resistant clones counted. Error bars represent one standard deviation around the mean. Wild-type cells are significantly less sensitive than BRCA1- or BRCA2-deficient cells (Student's t-test P < 0.05). c, Inhibition of PARP activity selectively inhibits the survival of cells lacking wild-type BRCA1 or BRCA2. Clonogenic survival curves of BRCA1 wild-type (11CO), heterozygous (Cre6) and deficient (Cre10) ES cells, and BRCA2 wild-type (D3), heterozygous (Cre15) and deficient (Cre24) ES cells after 10–12 days continuous exposure to chemical inhibitors. Error bars represent one standard deviation around the mean. d, Exposure to PARP inhibitor for 24 h results in G2/M arrest. ES cells were treated with 10 nM or 1 M KU0058684 or vehicle and analysed by FACS. e, Exposure to PARP inhibitor for 48 h results in apoptosis. ES cells were treated with KU0058684 or vehicle and stained with an annexin V antibody and propidium iodide (PI). The lower right quadrant of each panel represents early apoptotic cells and the upper right quadrant represents late apoptotic/necrotic cells. f, Chromosome analysis of BRCA1- and BRCA2-deficient ES cells exposed to KU0058684 or vehicle for 24 h. The small arrowheads indicate complex chromatid rearrangements and the large arrowhead indicates a chromatid break.