Crucial roles of MVP, GGT-II, and mutant p53 in Arf6 activation. (A–C) Arf6 activation (A), Matrigel invasion (B), and c-Met activation (C) upon TGFβ1 stimulation were measured using MDA-MB-231 cells preincubated with the indicated inhibitors or DMSO for 16 h. Matrigel invading cells were quantified as described in the Matrigel invasion assay section within Materials and methods (n = 3). (D) HGF-induced Arf6 activation was measured in MDA-MB-435s cells and Hs578T cells preincubated with Simvastatin or DMSO for 16 h. (E and F) Subcellular localization of Arf6-EGFP, expressed in MDA-MB-231 cells, and their p53 derivatives was examined before (−) and after (+) 5 min TGFβ1 stimulation, together with the visualization of F-actin using Alexa Fluor 647–conjugated phalloidin (E). Relative amounts of Arf6-EGFP localized at the cell periphery before and after the stimulation (F) were estimated from >10 cells as described in the Immunofluorescence microscopy section within Materials and methods (n = 2). (G–K) Ligand-induced Arf6 activation was measured in breast cancer cells, as indicated, and preincubated with or without 2 µM FTI-277 or GGTI-2133 for 16 h (G). TGFβ1-induced Arf6 activation (H), Matrigel invasion (I), and PM recruitment of Arf6 (J and K) were measured in MDA-MB-231 cells pretreated with siRNAs for GGT-I, GGT-II, or Irr, as indicated. Matrigel invasion activities were measured as in B (n = 3), and PM recruitment of Arf6 was estimated as in F (n = 2). Representative images are shown from two independent experiments in which >10 cells were examined in each experiment. Simvastatin + MVA/MVAP, cells incubated with mevalolactone and MVA 5-phosphate for 6 h before addition of Simvastatin. The results represent mean ± SEM. *, P < 0.001. Bars, 10 µm.