Atovaquone inhibits the mTOR pathway by inducing expression of REDD1, a TSC1/2-dependent negative regulator of mTOR kinase activity. (A) Schematic overview of the strategy to identify a gene expression signature associated with atovaquone (AQ) treatment. Validation was done in SK-BR-3, MDA-MB-468, INA-6, RPMI 8226, and MV4-11 cells. (B) K562 cells exposed to AQ (20 μM) or rapamycin (10 nM) for 5 hours were analyzed by western blot with the indicated antibodies. (C) AQ inhibits induction of pS6 across leukemia cell lines. Pretreatment with increasing concentrations of AQ for 5 hours demonstrates dose-dependent reductions in pS6 induced by 15 minutes of exposure to HS5 CM. (D) SKBR3 cells were transfected with control or REDD1-targeting siRNA for 48 hours, then treated with AQ (25 μM) for 4 hours, followed by western blotting with the indicated antibodies. (E) TSC2-null or REDD1-null MEFs, or MEFs from their respective littermate controls, were treated with AQ (25 μM) for 2.5 hours, followed by western blotting. All data are representative of at least 2 independent experiments.