Novel Inhibitor of Geranylgeranyltransferase-I cells were stained with phalloidin to observe actin cytoskeleton. As shown in Figure 5A, P61-E7 treatment caused disorganiza- tion of actin cytoskeleton and significant cell rounding. We next examined whether P61-E7 treatment had effects on focal adhesion. This was examined by using antibody against vinculin, one of the components of focal adhesion. As seen in Figure 5A, the treatment with P61-E7 led to the disappearance of vinculin punctate immunostaining at the periphery of Panc-1 cells. The staining became uniform throughout the cell. The GGTI treatment had no effect on vinculin expression level (data not shown). These results suggest that focal adhesion assembly was inhibited by P61-E7. In addition, a soft agar clonogenicity assay was used to measure the effect of P61-E7 on the anchorage-independent growth of Panc- 1 cells. Treatment with increasing concentrations of P61-E7 impaired the anchorage-independent growth of Panc-1 cells in a dose-dependent manner (Figure 5B and 5C). For examples, at 5 mM, P61-E7 inhibited colony formation by 42.9%64.1%, while it inhibited colony formation by 71.2%65.2% at 10 mM (Figure 5B). Stable Expression of Farnesylated RhoA in Panc-1 Cells Suppresses GGTI-induced Phenotypes RhoA, a geranylgeranylated small GTPase, has been shown to influence a number of cellular processes including actin stress fiber Figure 5. Effects of P61-E7 on cell morphology and anchorage-independent growth of Panc-1. (A) Panc-1 cells were serum-starved in the presence of DMSO or 5 mM P61-E7 for 24 h, followed by stimulation with 10% FBS in DMEM for 30 minutes. Actin fibers and focal adhesions were visualized with TRITC-phalloidin and mouse anti-vinculin followed by FITC-conjugated anti-mouse antibody, respectively. Data are representative of three independent experiments. (B) Cells were seeded at a cell density of 1000 cells/well in 12-well culture dishes in 0.3% agar over a 0.5% bottom agar layer. DMSO or various concentrations of P61-E7 were included in the top layer of cells. Cultures were re-fed and treated with the GGTI or DMSO once weekly (14 days of incubation in total). Colonies were stained with 1 mg/ml MTT for 1 hour and scanned. Shown are representative results from three independent experiments, each in duplicate. (C) P61-E6 inhibits anchorage-independent growth of Panc-1. Growth in soft agar relative to the DMSO control (100%) is plotted. Shown are the averages 6 S.D. of three independent experiments, each in duplicate. doi:10.1371/journal.pone.0026135.g005 PLoS ONE | www.plosone.org 7 October 2011 | Volume 6 | Issue 10 | e26135