Transfection Procedure and Creation of Stable Cell Lines Panc-1 cells were grown to 80% confluency on 6-well plates and transfected with LipofectamineTM 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. Briefly, 10 ml of LipofectamineTM 2000 reagent was diluted in 250 ml of OPTI- MEM medium (Invitrogen, Carlsbad, CA) and was allowed to equilibrate at room temperature for 5 min. 4.0 mg of plasmid DNA per 250 ml of medium was suspended and allowed to complex with the diluted LipofectamineTM 2000 for 20 min at room temperature. Transfection complexes were added to cells in serum-free DMEM without antibiotics and incubated at 37uC for 4–6 h. Medium was then changed and replaced with DMEM containing 10% FBS, with penicillin-streptomycin, and the cells were further incubated at 37uC for 48 h. To generate stable cell lines, cells were passaged at 1:10, 1:20, and 1:50 dilutions into selective medium containing 800 mg/ml of GeneticinH (G418 sulfate; Invitrogen, Carlsbad, CA). Cell clones were expanded into mass culture, and expression of 3xHA-RhoA-GG and 3xHA- RhoA-F was analyzed by Western blotting using antibodies against the HA tag. Subcellular Fractionation NIH3T3 cells and Panc-1 cells stably expressing 3xHA-RhoA were treated with DMSO, P61-A6, or P61-E7 for 48 h. Cells were then washed and scraped into phosphate-buffered saline (PBS) and centrifuged at 2,500 rpm for 5 min. Pellets were resuspended (10 mM HEPES/KOH at pH 7.3, 10 mM KCl, 5 mM MgCl2, 0.5 mM DTT, and 16 protease inhibitor mixture), incubated on ice for 30 min and homogenized. Homogenates were centrifuged at 10006g for 10 min to collect the cytosolic fractions (superna- tant). The remaining pellets were then resuspended in buffer containing 1% Triton X-100, 150 mM NaCl, 20 mM Tris-HCl at pH 7.5, 1 mM EDTA, and 16 protease inhibitor mixture, and centrifuged at 15, 000 rpm for 15 min to collect the membrane- containing fractions (supernatant). Na+/K+ ATPase-a and RhoGDI were used as markers for the membrane-containing fractions and the cytosolic fractions, respectively. Western Blotting Cells were treated with DMSO, FTI BMS-225975, GGTI-298, P61-A6 or P61-E7 for 48 h, harvested, and lysed in lysis buffer (1% Triton X-100, 150 mM NaCl, 20 mM Tris-HCl at pH 7.5, 1 mM EDTA, and 16 protease inhibitor mixture). Proteins were then resolved by 12% or 12.5% SDS-PAGE and immunoblotted with antibodies against unprenylated form of Rap1 (U-Rap1; Santa Cruz Biotechnology, Inc.), p21CIP1/WAF1 (Millipore, Teme- cula, CA), p27Kip1 (rabbit, Santa Cruz Biotechnology, Inc.), phospho-p27Kip1 (T187) (Invitrogen, Carlsbad, CA), HDJ-2 (NeoMarkers, Fremont, CA), RhoGDI (Santa Cruz Biotechnolo- gy, Inc.), Na+/K + ATPase-a (Sigma), RalA (BD Bioscience), HA.11 (Covance) and Actin (Calbiochem). Detection was performed using peroxidase-conjugated secondary antibodies (Biorad) and Amersham ECL PlusTM Western Blotting Detection Reagents (GE Healthcare Life Sciences). Select bands were quantified using ImageJ imaging processing program (National Institutes of Health). Immunoprecipitation of p27Kip1 Protein Panc-1 cells were lysed in lysis buffer (1% Triton X-100, 150 mM NaCl, 20 mM Tris-HCl, pH 7.5, 1 mM EDTA, and 16 protease inhibitor mixture). Lysates were incubated with p27Kip1 antibodies (rabbit, Santa Cruz Biotechnology, Inc.) in 50% Protein G/Sepharose beads slurry for 2 h at 4uC while rocking, then Novel Inhibitor of Geranylgeranyltransferase-I washed four times with an excess of lysis buffer. Samples were then boiled at 95uC for 10 min in 46 sodium dodecyl sulfate (SDS)- sample buffer and analyzed by Western blotting as described above. Cell Proliferation Assays and Cell Cycle Analyses Effects of GGTIs on cell proliferation were examined using the CCK-8 cell counting kit (Dojindo Molecular Technologies, Kumamoto, Japan) as described previously [20]. Briefly, cells (36103) were seeded onto 96-well plates. The following day cells were treated with the appropriate inhibitor as indicated in the figure legends under low-serum conditions (0.5% FBS) for 72 hours. Cell Proliferation was calculated relative to the DMSO control. The cell cycle profiles were analyzed by flow cytometry as described previously [32]. Fluorescence Microscopy To examine the effects of GGTI on actin cytoskeleton and focal adhesion formation, cells were seeded on 4-well or 8-well chamber slides. The following day, cells were treated as indicated in the figure legends. Then cells were fixed in 3.7% formaldehyde-PBS and permeabilized in 0.1% Trion X-100-PBS for actin fiber and vinculin detection. Actin fibers were detected by incubation with fluorescein isothiocyanate (FITC)-labeled phalloidin or tetra- methyl rhodamine isothiocyanate (TRITC)-labeled phalloidin (Sigma-Aldrich, St. Louis, MO) as indicated in figure legends. Vinculin was detected with anti-vinculin antibodies (mouse, Sigma-Aldrich, St. Louis, MO) and fluorescently conjugated anti-mouse IgG antibody (Sigma-Aldrich, St. Louis, MO). Cell images were visualized using a Zeiss Microscope (406 magnifica- tion). Images were captured using AxioVision software. Anchorage Independent Growth Assay Cells were seeded at a cell density of 1000 cells/well in duplicate in 12-well culture dishes in 0.3% agar over a 0.5% bottom agar layer. Various concentrations of P61-E7 or DMSO were incubated 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 (tetrazolium salt) for 1 hour and scanned. Results Synthesis of GGTI P61-E7 by the Derivatization of the Carboxyl Group on the Dihydropyrrole Ring of GGTI P3- E5 We previously reported a novel small molecule GGTI compound P61-A6 (Figure 1A) [20]. This compound was identified by screening a pilot library of allenoate-derived compounds made through phosphine catalysis for scaffolds that can be used for further derivatization. The dihydropyrrole scaffold identified was then derivatized to identify compounds with improved potency and bioavailability. A particularly useful observation was made concerning a modification of the carboxyl group off of the dihydropyrrole ring that resulted in a dramatic increase in cellular potency [19,20]. Along the way, we succeeded in establishing a novel chemical library of .4,000 compounds through phosphine-catalyzed annulations of resin-bound alleno- ates. The above initial study identified another scaffold that consists of a tetrahydropyridine ring. P3-E5, a representative tetrahydro- pyridine carboxylic acid compound, inhibits GGTase-I with IC50 value of 0.31 mM [20]. The inhibition was specific to GGTase-I, as PLoS ONE | www.plosone.org 3 October 2011 | Volume 6 | Issue 10 | e26135