PARP inhibition causes a decrease in Alt-EJ that is enhanced by Ku70 loss.

<p>(<b>A</b>) PARPi treatment (Olaparib) causes a specific decrease in Alt-EJ, compared to Distal EJ. Shown are the immunoblot signals for PARylated protein and actin after treatment of U2OS cells with 5 μM of the PARPi Olaparib (2 days). Also shown is the cell cycle profile of siCTRL treated U2OS cells incubated with 5μM Olaparib (Olap), or mock treated (UN) prior to BrdU labeling and PI staining as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004943#pgen.1004943.g002" target="_blank">Fig. 2C</a>. The U2OS reporter cell lines described above were transfected with the I-SceI expression vector and treated with 5 μM Olaparib (Olap) or vehicle (DMSO). Shown are the %GFP+ cells for each reporter relative to vehicle treatment (DMSO). *<i>P</i><0.0001 distinct from the effect on Distal-EJ (<i>n</i> = 13). (<b>B</b>) Effect of PARPi on Alt-EJ is approximately additive with loss of FANCA. Shown are GFP+ frequencies for U2OS cells treated with an siRNA targeting FANCA (siFANCA-3) or siCTRL, and subsequently examined as in A, relative to siCTRL/DMSO treated cells. *<i>P</i><0.0001 distinct from the effect on Distal-EJ (n≥8). (<b>C</b>) Loss of Ku70 enhances the effect of PARPi on Alt-EJ in mES cells. Shown are immunoblot signals for PARylated protein and actin for WT, <i>Fanca^−/−</i>, <i>Ku70^−/−</i> and <i>Fanca^−/−Ku70^−/−</i> mES cells treated with Olaparib (2 μM, 2 days) or vehicle (DMSO). *indicates nonspecific band. Each of these mES cells with the EJ2-GFP reporter were transfected with I-SceI and treated with 2 μM Olaparib (Olap) or vehicle (DMSO). Shown is the frequency of %GFP+ cells relative to vehicle treated (DMSO). *<i>P</i>≤0.001 (<i>n</i>≥9).