Apoptotic resistance in spheroids is not due to the PI3K/Akt/ mTOR pathway. (A) The PI3K/Akt/mTOR pathway is inhibited by rapamycin and PI-103 in A549 monolayers and spheroids. A549 monolayers and spheroids were exposed to nothing, rapamycin (25 nM), or PI-103 (1 μM) for 4 hours, and then studied by immunoblot for expression of p-Akt (Ser473) and p-S6K (Thr389). Rapamycin inhibited mTOR activity, as shown by a decrease in the mTOR target, p-S6K, and PI-103 inhibited both PI3K and mTOR, as shown by a decrease in p-Akt and p-S6K. (B) PI3K/Akt/mTOR inhibitors do not reduce apoptotic resistance of spheroids to bortezomib plus TRAIL. A549 monolayers (open bars) and spheroids (solid bars) were treated with 100 nM bortezomib (Bz) plus 1 ng/ml TRAIL with either no inhibitor, 25 nM rapamycin, or 1 μM PI-103 for 24 hours. The inhibitors did not reduce the apoptotic resistance of spheroids to bortezomib plus TRAIL (*different from monolayer; mean ± SD; n = 3; P < 0.001).