Figure 1. PI 3-K inhibition and depletion lead to AKT reactivation.
(A) MDA-MB-468 cells were treated with BKM-120 (1 or 5 μM), MK-2206 (10 μM) for 3 or 48 hours, alone or with 1 μM of BKM-120, BYL-719, or MK-2206 for an additional 3 hours, as indicated. Cells were then harvested and lysates were immunoblotted for the indicated total and phosphorylated proteins. T, Thr; S, Ser.
(B) MDA-MB-468 cells were infected with shRNA lentivirus targeting PIK3CA or control vector (pLKO). After selection, cells were harvested, and lysates were immunoblotted for the indicated total and phosphorylated proteins.
(C) MDA-MB-231 cells were infected with shRNA lentivirus targeting PIK3CA or control pLKO. Cells were serum-starved overnight (−) or stimulated with IGF-1 for 20 min (+), harvested, and lysates were immunoblotted for the indicated antibodies.
(D) MDA-MB-231 cells were infected with shRNA lentivirus targeting PIK3CA, PIK3CB, PIK3CD, PIK3CG or control pLKO. Cells were serum-starved overnight (−) or stimulated with IGF-1 for 20 min (+), harvested and lysates were immunoblotted with the indicated antibodies. Blots are representative of at least 3 independent experiments. See also fig. S1A–C for additional experiments with BKM-120 time course, depletion of PIK3CA with multiple shRNA hairpins and analysis of pAKT1 pS473 and pS474 with PIK3CA depletion.