Fig. 4.

PI3K-inhibition increases poly-ADP-ribosylation and H2AX phosphorylation. A. Compensatory pathway activation induced by treatments with NVP-BKM120. HCC1937 or SUM149 cells were treated with NVP-BKM120, Olaparib or its combination as indicated for 72 hours, lysed and subjected to immunoblotting with antibodies against total AKT, EGFR, ERK and their phospho-specific epitopes. B. In vivo increase of γH2AX-positive cells after treatment with NVP-BKM120 and proliferative activity at the “pushing margin”. Tumor-bearing mice were subjected to a pre-treatment biopsy and then treated with NVP-BKM120 at 50 mg/kg/day. IHCs of pre-treatment biopsies and post-treatment tumor tissues were performed with antibodies as indicated. C. Effects of combined PI3K- and PARP-inhibition on BRCA1-mutant cells. Cells were treated with NVP-BKM120 at 1 µM and Olaparib 10 µM or their combination for 24 hours, lysed and subjected to immunoblotting with antibodies against PAR, pAKT, total AKT and γH2AX and Actin as indicated. D. BRCA1 mutant human HCC1937 or SUM149 cells were treated with vehicle control or NVP-BKM120 at the indicated concentrations for 24 hours, lysed and subjected to immunoblotting with antibodies against PAR, p-AKT (S473), γH2AX, Cleaved Caspase 3(CC3) as an apoptosis marker and Actin as a loading control.