according to the manufacturer’s protocol.). Briefly, after TZD treatment, cells were treated with 100 mL of MTS/PMS solution from Promega’s CellTiter 96 Aqueous one solution cell prolifer- ation assay for 4 hours. Proliferation was measured by colorimetric absorbance at 492 nm. The incorporation of BrdU into DNA was assessed using a Cell proliferation, ELISA BrdU kit (Roche). In brief, cells were labeled after culture with BrdU labeling solution overnight. Afterwards, cells were fixed, the DNA denatured, and peroxidase the BrdU complex was then detected and quantitated colorimetrically at 450 nm. Treatments were normalized to control (DMSO vehicle treatment) and expressed as the relative fold change compared to the control. BrdU proliferation was also preformed on cells that were transfected with or without a dominant negative (DN) or overexpressing (OE) PPARc vector. Cells were double transfected as described under the constructs and transfection section below, then serum starved for 24 hours and treated for 24 hours prior to running a BrdU assay in combination with either the agonists or antagonists. Cell Cycle assay Flow cytometry was used to determine the effect of TZD treatment on cell cycle kinetics, as previously described by Vindelov [53]. Cells were cultured until they were 60–90% confluent and treated as described above. Briefly, cells were washed with PBS, trypsinized for 5–10 minutes and then treated with trypsin inhibitor and ribonuclease A for 30 minutes. Subsequently, propidium iodide (final concentration 50 ug/mL) in combination with sperimine HCl was added to the cells for 30 minutes in the dark at 4uC. The suspension was analyzed using a FacsCalibur flow cytometer from Becton Dickson (San Jose, CA) at the core facility at the University of Kentucky using Mod FitLT V.3.1 software. Ratios of cells in the G0/G1, S, and G2/M phases of cell cycle were determined on the basis of their DNA content and presented as cell percentage at the end stage of the cell cycle. Cell cycle histograms were obtained from 3 determinations, each with a total of 100,000 cells/treatment. Cell cycle was also performed on cells that were double transfected with either DN or OE PPARc. Cells were double transfected as outlined under the constructs and transfection section below, then serum starved for 24 hours and treated for 24 hours. Consequently, a cell cycle assay was performed. Apoptosis assay Cells were grown as described above and treated for 4, 8 or 12 hours with one of the following: Vehicle control (0.1% DMSO), or 10 mM of CGZ or TGZ. Cells were then analyzed for apoptosis using the Vybrant Apoptosis Assay kit per the manufacturer’s protocol (Invitrogen). Briefly, cells were washed twice with PBS, trypsinized and centrifuged. Annexin V Binding buffer was used to resuspend the pellet and Alexa Flour 488 annexin V and propidium iodide solution was added to the cells and incubated at room temperature for 15 minutes. Samples were analyzed using flow cytometry readings at 530 nm and .575 nm in the FacsCalibur flow cytometer at the core facility at the University of Kentucky. Constructs and transfection assays PPARc regulates gene expression by binding to a specific Peroxisome Proliferator Response Element (PPRE) [54]. To References PPARc Ligands Differentially Impact OVCAR Cells determine whether TZD treatments result in the activation of the PPARc promoter, PPRE reporter constructs were utilized. This construct, 3XPPRE-TK-pGL3, contains three copies of the PPRE sequence (AGGACAAAGGTCA). The DN construct was a kind gift from Drs. O’Rahilly and Chatterjee (Cambridge University, U.K.) and has been modified to introduce a stop codon at amino acid 462 and has been previously characterized and used by our lab [19]. The wild type overexpression (OE) PPARc construct was made by our laboratory. The luciferase construct lacking the PPRE and the Renilla constructs were purchased from Promega. Approximately 120,000 cells (about 30% confluence) were plated per well for all transfection experiments. To compare the PPRE activity across the three different cell lines, cells were either transfected with a 3XPPRE mTK-Luc or with two vectors (a luciferase lacking PPRE and a Renilla containing vector) [55]. Cells were transfected two subsequent days and then lysed on day 3 for analysis. To inspect the effect of TZD treatment on PPRE activity, cells were transiently double transfected with a total of 0.4 mg DNA per well. Each well was transfected with 0.2 mg of pGL3 plasmid containing 3XPPRE mTK-Luc and Renilla [55] using FuGENE transfection reagent (Roche). Cells were also transfected with 0.2 mg of one of the following: Empty vector control construct, DN, or wild type overexpression (OE) PPARc. Cell transfection was repeated for an additional 24 hours. Afterwards, cells were treated with 10 mM of one of the following: Vehicle control (DMSO), Rosi, CGZ, TGZ, Pio or 9-cis-RA for an additional 24 hours. Cells were lysed with 150 ml of passive lysis buffer and treated according to the manufacturer’s instructions (Promega dual luciferase assay kit, Promega). A total of 20 ml was used for luminometry. Luminometry was performed on a Berthold Technologies Lumat 9507 (Willbad, Germany). Ratios between luciferase and Renilla were used to calculate promoter activity and adjust for differences in transfection efficiency. Transfection efficiencies were calculated and revealed that the highest transfection efficiency was achieved in Skov3 cells followed by Ovcar3 with the CaOv3 cells having the lowest transfection efficiency. Data is presented as a relative expression normalized to the vehicle control. Statistical analysis All data are presented as means 6 SEM. One-way analysis of variance (ANOVA) or Welch’s t-test was used to test differences among treatments. If the ANOVA showed significant effects, post hoc tests were performed using Tukey’s or Bonferroni, in order to identify significant differences among treatments. The means were compared with p#0.05 considered significant. Statistical analysis was performed using a statistical analysis software [56]. Acknowledgments We would like to thank Dr. Misung Jo, Dr. Yekaterina Zaytseva and Natalie Wallis for their critical evaluation and review of our work. Author Contributions Conceived and designed the experiments: LFA RCS MWK TEC. Performed the experiments: LFA RCS MWK TEC. Analyzed the data: LFA RCS MWK TEC. Contributed reagents/materials/analysis tools: LFA RCS MWK TEC. Wrote the paper: LFA RCS MWK TEC. 1. Choi JH, Wong AS, Huang HF, Leung PC (2007) Gonadotropins and ovarian cancer. Endocr Rev 28: 440–461. 2. Zhang GY, Ahmed N, Riley C, Oliva K, Barker G, et al. (2005) Enhanced expression of peroxisome proliferator-activated receptor gamma in epithelial ovarian carcinoma. Br J Cancer 92: 113–119. 3. Vanderhyden BC, Shaw TJ, Ethier JF (2003) Animal models of ovarian cancer. Reprod Biol Endocrinol 1: 67. 4. Flessner MF, Choi J, He Z, Credit K (2004) Physiological characterization of human ovarian cancer cells in a rat model of intraperitoneal antineoplastic therapy. J Appl Physiol 97: 1518–1526. PLoS ONE | www.plosone.org 13 January 2011 | Volume 6 | Issue 1 | e16179