BEZ235-induced apoptosis is associated with suppression of mTORC1 and DDR-regulated substrates, but not inhibition of PI3K/mTORC2. (A) Eμ-Myc (#4242) lymphoma cells were treated with Everolimus, BEZ235 or vehicle (DMSO) at the concentrations indicated for 1 hour prior to harvesting and preparation of protein lysates. Protein was then separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) prior to immunoblotting with phospho-specific antibodies for mTORC1-regulated substrates as indicated. Total protein and actin or tubulin loading controls are provided for each blot. (B) Lysates from the same treatment conditions were blotted for mTORC2- (phospho-Ser473 AKT) and PI3K- (phospho-Ser241 PDK-1 and Tyr223 BTK) regulated targets as indicated. (C) Eμ-Myc (#4242) lymphoma cells were treated with Everolimus, BEZ235, or vehicle (DMSO) at the concentrations indicated for 1 hour prior to harvesting and preparation of protein lysates, separation by SDS-PAGE, and immunoblotting for phospho-RPS6, γH2AX, and β-actin as indicated. (D) Eμ-Myc #4242 MSCV/Bcl2 lymphoma, either nonirradiated or irradiated (6 Gy), prior to 30-minute treatment with BEZ235 (500 nM), KU55933 (4 μM), or vehicle (DMSO) was harvested, separated by SDS-PAGE, and immunoblotted for DDR-kinase and mTOR-regulated phosphorylation substrates as indicated. Control samples were obtained from nonirradiated p53 mutant (#106) and p53 null (#3239) lymphomas. Each western blot is representative of at least 3 independent experiments.