Expression profiling of genes induced by atovaquone treatment

The drug atovaquone inhibits the mTOR pathway, as identified by western blotting to phospho-S6 and phospho-4E-BP1. However, this effect only occurs at longer time points (2-5 hours) and is blocked by cycloheximide or actinomycin D, demonstrating that de novo gene expression is required. To identify the factor upregulated by this drug, we performed gene expression profiling in two separate cell lines that respond to atovaquone. U266 cells were treated with vehicle (DMSO) or atovaquone (15 micromolar) for 2 hours. K562 cells were treated with vehicle or atovaquone (20 micromolar) for 5 hours. RNA was harvested by Trizol, further purified by RNeasy spin column, and then submitted to the Dana-Farber Cancer Institute Molecular Diagnostics Laboratory.