Pioglitazone inhibits aromatase expression in human preadipocytes. Pioglitazone inhibits aromatase expression in human preadipocytes. A, cells were transfected with 1.8 μg PPRE-luciferase and 0.2 μg pSVβgal. Following transfection, cells were treated with the indicated concentrations of pioglitazone for 24 hours. In A, D, and E, cells were lysed and luciferase activity was measured in cell lysates. Firefly luciferase activity was normalized to β-galactosidase activity. B, cells were treated with the indicated concentration of pioglitazone for 24 hours and then aromatase activity was assayed in cell lysates. Enzyme activity is expressed as femtomoles/μg protein/min. C, total RNA was prepared from cells that had been treated with pioglitazone for 24 hours. Levels of aromatase mRNA were quantified by real-time PCR. Values were normalized to levels of β-actin. D, cells were transfected with 1.8 μg CYP19 I.3/II promoter and 0.2 μg pSVβgal. Cells were then treated with indicated concentrations of pioglitazone for 24 hours. E, cells were transfected with 0.9 μg CYP19 I.3/II promoter and 0.2 μg pSVβgal. Cells also received 0.9 μg siRNA to GFP (control siRNA) or PPARγ. 24 hours after transfection, cells were treated with vehicle or 10 μmol/L pioglitazone. Aromatase promoter activity was measured 24 hours after treatment. In the inset, Western blotting was conducted on cell lysates that were treated with siRNA to GFP (control siRNA) or PPARγ. The blot was probed with antibodies to PPARγ and β-actin. F, cells were treated with indicated concentrations of PGE2. 24 hours later, aromatase activity was determined. G, cells were treated with indicated concentrations of pioglitazone for 16 hours. Subsequently, the concentration of PGE2 in the cell culture medium was measured using enzyme immunoassay. In H and I, cells were treated with pioglitazone for 18 hours. Subsequently, cellular levels of cAMP (H) and PKA activity (I) were determined. A–I, mean ± SD are shown, n = 6. *, P < 0.01 compared with vehicle-treated cells. Kotha Subbaramaiah et al. Cancer Prev Res 2012;5:1183-1194 ©2012 by American Association for Cancer Research