Figure 8. Stable expression of farnesyalted RhoA rescues cells from cell rounding and inhibition of focal adhesion assembly caused by P61-E7. Panc-1 cells, Panc-1 cells stably expressing 3xHA- RhoA-GG wild-type, and Panc-1 cells stably expressing 3xHA-RhoA-F mutant were serum-starved in the presence of DMSO or 5 mM P61-E7 for 48 h, followed by stimulation with 10% FBS in DMEM. Actin fibers and focal adhesions were visualized with FITC-phalloidin and mouse anti-vinculin followed by TRITC-conjugated anti-mouse antibody, respectively. Data are representative of three independent experiments. doi:10.1371/journal.pone.0026135.g008 Novel Inhibitor of Geranylgeranyltransferase-I involved in the GGTI effects on fibroblast cells [45]. Further work is needed to express mutant forms of these proteins that can bypass geranylgeranylation to gain complete understanding of the mechanism of GGTI effects. Supporting Information Figure S1 Effects of P3-E5 on the enzymatic activities of GGTase-I, RabGGTase, and FTase. GGTase-I and FTase activities were determined by following the incorporation of radiolabeled isoprenoid [ 3 H]geranylgeranyl or [3H]farnesyl into substrate proteins. FTase or GGTase-I (50 nM) was used to initiate reactions containing 0.4 mM [3H]farnesyl diphosphate (FPP) (21.5 Ci/mmol, Perkin Elmer Life Sciences) or 0.5 mM 3 [ H]geranylgeranyl diphosphate (GGPP) (23.0 Ci/mmol, Perkin Elmer Life Sciences) and 2 mM maltose-binding protein-tagged substrates (K-Ras4B for FTase and RhoA for GGTase-I) in 20 ml of buffer containing 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 5 mM ZnCl2, 0.01% Triton X-100, and 1 mM dithiothreitol. Inhibitors were added at the indicated concentrations. The final DMSO concentration was 2.5% for all samples. Reactions were carried out for 10 min at 30uC. The reaction mixture was spotted onto a filter paper and treated with 10% trichloroacetic acid, followed by ethanol and acetone washing. The filter was counted using a scintillation counter. For RabGGTase assays, the reaction contained the following components in 20 ml: 0.625 ml of [3H]GGPP (0.7 mM), 25 nM RabGGTase, 0.6 mM REP-1, 0.6 mM purified Rab7 or Ypt1 protein, 40 mM HEPES (pH 7.5), 150 mM NaCl, 5 mM dithiothreitol, 3 mM MgCl2, and 0.3% CHAPS. Reactions were carried out for 20 min at 37uC, and the products were analyzed as described above for the GGTase-I reaction. Data represent the mean 6 S.D. of two measurements from two independent experiments. (TIF) Figure S2 Effects of P61-E7 on p27Kip1 protein levels. (A) Panc-1 cells were treated with DMSO or various concentra- tions of P61-E7, and whole cell lysates were collected and resolved on SDS-PAGE for immunoblotting using antibodies against p27Kip1. Results shown are representative of three independent experiments. (B) and (C) Panc-1 cells were treated with DMSO or P61-E7 (5 mM) for 48 h. Whole cell lysates were collected and were immunoprecipitated (B) with anti-p27Kip1 antibodies in 50% Protein G/Sepharose beads slurry (negative control for immuno- precipitation: whole cell lysates from untreated Panc-1 cells mixed with slurry, without antibodies). (B) Immunoprecipitates were then resolved on SDS-PAGE for immunoblot analysis using phospho- 27Kip1(T187) antibodies (B, top panel) or p27Kip1 antibodies (B, bottom panel). Lanes: 1, DMSO-treated; 2, P61-E7-treated; 3, No antibody control. (C) Remaining lysates (10 mg) from each sample were resolved on SDS-PAGE for immunoblotting using antibodies against p27Kip1 (C, top panel) or actin (C, bottom panel) to determine total p27Kip1 level in each input used for immunopre- cipitation. Results shown are representative of three independent experiments. The RhoGDI, Actin, phospho-p27Kip1 and total p27Kip1 bands were quantified using ImageJ, and the results are given above the images as fold change compared to the DMSO control. (TIF) Figure S3 P61-E7 inhibits RhoA activation in Panc-1 cells. Panc-1 cells were transfected with 3xHA-RhoA pcDNA expression vector. Twenty-four hours after transfection, cells were serum-starved in the presence of DMSO or P61-E7 for 24 h. Then, cells were stimulated with 10% FBS in DMEM in the PLoS ONE | www.plosone.org 11 October 2011 | Volume 6 | Issue 10 | e26135