investigate the effect of the four different TZDs on ovarian cancer cells and determine whether these effects are PPARc dependent or independent. Results Ovcar3, CaOv3 and Skov3 ovarian cancer cell lines express PPARc In order to determine whether ovarian cancer cells express PPARc, real time PCR and western blot analysis was performed. There was differential PPARc expression in the three different cell lines both at the mRNA and protein levels. While PPARc mRNA expression was highest in Skov3 cells (Figure 1A), Skov3 cells had the lowest PPARc protein levels (Figure 1B). In contrast, Ovcar3 had low levels of PPARc mRNA expression but abundant expression of PPARc protein (Figures 1A and 1B respectively). PPARc activity in the three cell lines was examined using cells transfected with a 3XPPRE-Luc-Renilla construct and compared to cells transfected with luciferase and Renilla constructs lacking the PPRE. Ovcar 3 cells exhibited approximately 2 fold more endogenous PPRE activity compared to CaOv3 cells, while Skov3 cells showed 50% more PPRE activity compared to Ovcar3 cells (Figure 1C). Retinoic Acid Receptor (RXR) is not a limiting factor in PPARc activity PPARc binds to its heterodimeric partner RXR before binding to DNA [16]. RXR is known to be constitutively expressed in cells and has been shown to heterodimerize with other receptors besides PPARc, such as the vitamin D receptor [17]. For RXR to be activated, its ligand 9-cis-retinoic acid (9-Cis-RA) needs to be present. In order to insure that activated RXR is not a limiting factor in our experiments, cells were treated with 9-Cis-RA. In these experiments, cells were transfected with different PPARc constructs including D467 which is a dominant negative (DN) form of PPARc [18,19] as well as an over expression form of wild type PPARc (OE) which is used to increase PPARc expression. A DN form of PPARc was used throughout these studies based upon initial experiments that revealed that DN transfection decreased the PPRE activity 40% below levels in cells transfected with ShRNA PPARc (data not shown). In order to insure that PPARc was overexpressed whether in the DN or OE form compared to control cells (i.e. cells transfected with PGL3), PPARc protein expression was measured using western blot analysis. As expected there was a marked induction of both the DN and OE form of PPARc (Figure 2A insert). After transfection with the control, DN or OE vector, cells were subsequently treated with vehicle control or 1 mM of 9-Cis-RA for 24 hours in the dark. We observed that the presence of 9-Cis-RA did not affect the activity of the PPARc reporter assay in Ovcar 3 cells, indicating that activated RXR is not a limiting factor in this cell line (Figure 2A). The addition of 9- Cis-RA to CaOv3 cells did not change PPRE activity until PPARc was overexpressed (Figure 2B) suggesting that activated RXR is not rate limiting until PPARc is highly abundant in this cell line. Surprisingly, in Skov3 cells, 9-Cis-RA increased the PPARc reporter activity assay in control cells (PGL3) but had no effect when PPARc was over expressed compared to control (Figure 2C). CGZ and TGZ cause a decrease in cell proliferation To define the effects of PPARc ligands on cell proliferation, MTS assays were performed. Ovcar3 cells were treated with concentra- tions of 0, 0.1, 1 or 10 mM of Rosi, CGZ, TGZ or Pio for 24 hours. The maximum TZD concentration used was 10 mM since PPARc specific actions are reported with TZDs at concentrations #10 mM [20] and higher concentrations are known to induce cell death [21]. PPARc Ligands Differentially Impact OVCAR Cells Figure 1. Expression of PPARc in three ovarian cancer cell lines (Ovcar3, CaOv3, Skov3). (A) mRNA expression (B) Protein expression and (C) PPRE luciferase activity. Data was normalized to levels of PPARc expression and activity in Ovcar3 cells. Results are the means 6 SEM for at least 3 measurements from three individual experiments. Bars that do not share a letter designation are significantly different (p,0.05). Cells transfected with Luc and Renilla constructs lacking PPRE (PPRE 2) were significantly lower than the same cell line transfected with PPRE- Luciferase-Renilla constructs (PPRE +) by Welch’s t-test (p,0.05). doi:10.1371/journal.pone.0016179.g001 Administration of the four TZDs resulted in differences in cell proliferation. Treatment with CGZ decreased proliferation 80%, TGZ caused a 65% decrease, while Rosi and Pio had no effect on proliferation (Figure 3A–D Black bars). In an effort to understand whether the decrease in proliferation following TZD treatment is common across other ovarian cancer cells, TZD treatment of CaOv3 and Skov3 was explored. A similar decrease in proliferation was seen in these cell lines when treated with CGZ and TGZ at PLoS ONE | www.plosone.org 2 January 2011 | Volume 6 | Issue 1 | e16179