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. 2012 Dec 18;7(12):e51746. doi: 10.1371/journal.pone.0051746

Figure 5. Selective antagonist GW9662 of PPARγ2 pro-adipocytic activity increases β-catenin protein stability.

Figure 5

U-33/γ2 cells were treated with either vehicle, 1 µM Rosi, 10 µM GW9662, or in combination for 72 h. A. Adipocyte differentiation was assessed by measuring the number of Oil Red O positive cells. B – C. Relative expression of adipocyte-specific gene markers. D. Osteoblast differentiation was assessed by measuring alkaline phosphatase activity. E – G. Relative expression of osteoblast-specific gene markers and Wnt10b. Fold change in transcript levels was calculated as compared to vehicle treated cells. H. Immunocytochemistry of β-catenin protein. Green: β-catenin; purple: DAPI staining of nuclei. I. Percentage of β-catenin positive cells (T) and cells positive for β-catenin in the nucleus (N). J. Western blot analysis of total β-catenin protein levels. Each lane was loaded with 50 µg of total protein lysate. K. Transcriptional activity of β-catenin measured with luciferase gene reporter assay using TOP-Flash construct. Promoter activity of firefly luciferase was normalized to renilla luciferase which was used as a transfection control. Statistically significant differences are shown between Rosi-treated samples and samples receiving combined treatment (* p<0.05; NS – non-significant). V – vehicle; R – Rosi; G – GW9662; GR – GW9662+ Rosi.