U87, A549, and MDA-MB231 were treated with simvastatin (10 μM, 12, 24, 36 hrs) and the abundance of RhoA, cdc42, and Rac1/2/3 in membrane and cytosolic fractions obtained from U251 (A), A549 (C), and MDA-MB-231 (E). GAPDH and Pan-Cadherin abundance was also assessed to control for loading in cytosolic and membrane fractions, and to confirm lack of cytosolic contamination in membrane fractions. Data are typical of 3 independent experiments using different primary cultures. Cropped representative of blots have been showed. G-LISA assay was done to evaluate the GTP-bound Rho protein in U251, A549, and MDA-MB231. Different conditions were tested for 36 hrs including starvation, Simva. (10 μM), Mev (2.5 mM), FPP, and GGPP (15 μM), Simva. + Mev, Simva. + FPP, and Simva. + GGPP. Simva. significantly increased GTP-bound Rho in U251 cells (B) while Mev, FPP, and GGPP co-treatment decreased GTP-bound Rho compared to control. none of the treatments significantly change GTP-bound protein in A549 (D) and MDA-MB231 (F) cells. For each experiment a positive control provided in the kit was used and control cells were treated with the reagent in the kit. Results are expressed as mean ± SD of 2 replicates in an independent experiment (***P < 0.001).