Figure 5
Suppression of BRCA2 levels and increased sensitivity to PARP inhibitors under mitochondrial dysfunction involves increased cytosolic calcium concentration. (a) BRCA2, Skp2 and miR-1245 levels were evaluated in total protein extracts or miRNA preparations of PNT1A cells cultured 6 h in the presence or absence of rotenone (350 nM), antimycin A (150 ng/ml), CCCP (2 μM) or solvent alone (Control). (b) Steady-state [Ca2+]i was measured using Fura-2AM. (c) PNT1A Rho(0) cells were treated with the calcium chelator BAPTA-AM for 1 h, incubated in fresh medium for 24 h, harvested and analyzed for BRCA2 and Skp2 protein levels by immunoblotting, and for miR-1245 levels by real-time quantitative PCR. (d) PNT1A wild-type cells were treated with the calcium ionophore ionomycin for 3 h, incubated in fresh medium for 24 h, harvested and analyzed for BRCA2 and Skp2 protein levels by immunoblotting, and for miR-1245 levels by real-time quantitative PCR. (e, f) Cells were treated with BAPTA-AM or ionomycin as described in (b) and (c), then added with fresh medium containing 10 μM AG014699. After 48 h, Rad51 foci formation (e) and apoptosis (f) were measured.
