as RhoA and RalA, known substrates of GGTase-I. NIH3T3 cells (Figure 3A and 3B) and Panc-1 cells stably expressing 3xHA-RhoA (Figure 3C and 3D) were treated with DMSO, P61-A6 or P61-E7, and membrane and cytosolic fractions were prepared and processed for SDS-PAGE followed by Western blotting. The Na+/K+ ATPase-a membrane marker and the RhoGDI cytosolic marker were used to confirm the separation of membrane- containing and cytosolic fractions, respectively. As shown in Figure 3, treatment with P61-E7 resulted in increases of RalA (Figure 3A and 3B) and 3xHA-RhoA (Figure 3C and 3D) in the cytosolic fractions of NIH3T3 cells and Panc-1cells stably expressing 3xHA-RhoA, respectively, while their association with the membrane fractions was decreased. Furthermore, at both concentrations tested (5 and 10 mM), P61-E7 is more effective at disrupting association of RalA (Figure 3A and 3B) and 3xHA- RhoA (Figure 3Cand 3D) with the membrane fractions when compared to P61-A6. P61-E7 is More Effective at Inhibiting Cell Cycle Progression, Inducing p21CIP1/WAF1 Protein Level and Blocking Cell Proliferation When Compared to P61-A6 As shown in Figure 4, treatment of Panc-1 (Figure 4A) and MiaPaCa-2 (Figure 4B) cells with either P61-A6 or P61-E7 for 48 h caused dose-dependent enrichment of G1 phase cells, whereas the Novel Inhibitor of Geranylgeranyltransferase-I percentage of S phase cells decreased. Consistent with findings discussed above, P61-E7 is more effective at inhibiting cell cycle progression in both cell lines tested (Figure 4A and 4B). For example, following treatment with P61-A6 (5 mM), the proportion of Panc-1 cells in the G1 phase increased from 45.1%63.1% (DMSO) to 48.4%61.4%, whereas the proportion of Panc-1 cells in the G1 phase increased to 62.7%62.2% following treatment with P61-E7 (5 mM) (Figure 4A). Likewise, at 5 mM, P61-A6 treatment caused an increase in proportion of MiaPaCa-2 cells in the G1 phase by 2.6%61.3% in comparison to DMSO treatment, while treatment of MiaPaCa-2 cells with 5 mM of P61-E7 increased the proportion of cells in the G1 phase by 9.7%60.67% when compared to DMSO treatment (Figure 4B). The accumulation of G1 phase cells was associated with an induction of proteins involved in the regulation of cell cycle. Both P61-A6 and P61-E7 induced p21CIP1/WAF1 expression level in Panc-1 (Figure 4C) and NIH3T3 (data not shown) cells in a dose- dependent manner. Similar to observations discussed above, P61- E7 treatment resulted in a higher induction of p21CIP1/WAF1 level when compared to treatment with P61-A6 in both cell lines tested. For example, at 20 mM, P61-A6 induced p21CIP1/WAF1 level by 25.9-fold in comparison to DMSO treatment, while treatment with 20 mM of P61-E7 induced p21CIP1/WAF1 level by 61.2-fold (Figure 4C). Figure 3. P61-E7 causes an increase of cytosolic RalA and RhoA in comparison to P61-A6. NIH3T3 cells (A,B) and Panc-1 cells stably expressing 3xHA-RhoA (C,D) were treated with DMSO, P61-A6, or P61-E7 for 48 h. Cytosolic and membrane fractions were prepared and processed for SDS-PAGE, followed by Western blotting using antibodies against RalA (A, top panel), the HA tag (C, top panel), Na+/K+ ATPase-a (A and C, middle panels) and RhoGDI (A and C, bottom panels). Data shown are representative of three independent experiments. The RalA (A, top panel), 3xHA-RhoA (C, top panel), Na+/K+ ATPase-a (A and C, middle panels) and RhoGDI (A and C, bottom panels) bands were quantified using ImageJ. Intensities of RalA and 3xHA-RhoA bands in the membrane or cytosolic fraction were normalized to their respective control for membrane or cytosolic fraction. Subcellular localization of RalA (B) and 3xHA-RhoA (D) are represented as Membrane/Cytosolic ratio, relative to the DMSO controls (which were set at a value of one). doi:10.1371/journal.pone.0026135.g003 PLoS ONE | www.plosone.org 5 October 2011 | Volume 6 | Issue 10 | e26135