Figure 6
Suppression of BRCA2 levels and inhibition of HR under mitochondrial dysfunction is dependent on a calcineurin/PI3-kinase/Akt pathway. (a) Wild-type and Rho(0) PNT1A cells were assessed for calcineurin, pAKT, total AKT and p85 levels by immunoblotting. (b–d) PNT1A Rho(0) cells were incubated for 1 h with 10 μM LY294002, 2 μM BAPTA-AM, 25 μM W-7, 10 nM FK506 or solvent alone (control), incubated for 24 h in fresh medium and then assessed for BRCA2, pAKT, total AKT and p85 levels by immunoblotting (b), for Skp2 and miR-1245 RNA levels by quantitative RT–PCR (c), and for spontaneous and rucaparib-induced Rad51 foci by immunofluorescence (d). (e) PNT1A Rho(0) cells were transiently transfected with AKT siRNA or non-specific siRNA (−) and after 48 h assessed for BRCA2, pAKT and AKT protein levels by immunoblotting (left) and for spontaneous Rad51 foci formation by immunofluorescence (right). (f) PNT1A wild-type cells were incubated for 6 h with the AMPK activator AICAR (0.5mM), incubated in fresh medium for 24 h, then analyzed for BRCA2 and Skp2 protein levels by immunoblotting.
