Enforced Akt activation promotes anoikis. A–C, MDA-MB-231 cells treated with DMSO, LY294002 (LY), or compound C (CC) were subjected to 48 hours of suspension and harvested for immunoblotting (A) and caspase-3 activity assay (B); n = 3. Treated cells were subjected to colony formation in methylcellulose for 15 days (C); n = 4. Error bars, mean ± SEM. D and E, Immunoblot analyses of MDA-MB-231 cells stably expressing control empty vector or HA myr-Akt (D), and control vector (expressing GFP) or GFP-tagged HA-Akt-T308D S473D (GFP Akt DD; E) cultured in adherent (Att) or suspension (Sus) condition; n = 3. F–H, After 48 hours of suspension, MDA-MB-231 cells stably expressing control vector (expressing GFP) or GFP Akt DD were subjected to immunoblotting for cleaved caspase-3 (F) and caspase-3 activity assay (G); n = 3. Cells were subjected to colony formation for 15 days (H); error bars, mean ± SEM of two experiments with three dishes each.