Atorvastatin-mediated disruption of prenylation and phosphorylation of signaling proteins. (A) Cellular cholesterol levels. MYC-expressing tumor cells were analyzed 24 hours after treatment. Equivalent numbers of cells were analyzed when MYC was expressed (MYC ON) or not expressed (MYC OFF) after doxycycline treatment (20 ng/mL) in the absence or presence of 10 μM atorvastatin (AT), 100 μM mevalonate (Mev), or 10 μM atorvastatin plus 100 μM mevalonate (AT + Mev). (B) Prenylation of several proteins was assessed by immunoblot when treated as described. Treatment with FTI-277 (5 μM) or GGTI-298 (5 μM) were used as controls to visualize changes observed in farnesylation and geranylation, respectively. (C) Ras protein accumulates in cytoplasm following atorvastatin treatment. MYC-induced lymphoma cell lines were treated for 6 hours with atorvastatin (AT; 10 mM). The cytoplasmic fraction was isolated and analyzed by Western blot for Ras protein. Representative data from 1 of 3 experiments is shown. (D) Kinetic analysis of changes in phosphoprotein expression analyzed by FACS analysis. MYC-induced lymphoma cell lines were treated for 24 and 36 hours, as indicated. The response to AT, Mev, and AT + Mev were determined and compared with the basal state, shown in the profile in black. Cells were analyzed for changes in phosphorylation of 56 different phosphoprotein epitopes. Changes observed for 19 of the epitopes are presented. (E) FACS plot analysis for changes in ERK1/2 phosphorylation. (F) Analysis of levels of protein expression and phosphorylation. Levels of ERK1/2, phosphorylated ERK1/2 (pERK1/2), Akt, and phosphorylated Akt (pS473) (pAkt) are measured by Western analysis.