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. Author manuscript; available in PMC: 2013 Jun 27.
Published in final edited form as: Cancer Prev Res (Phila). 2012 Jul 10;5(10):1183–1194. doi: 10.1158/1940-6207.CAPR-12-0201

Figure 4.

Figure 4

Pioglitazone modulates binding of pCREB, BRCA1, and p300 to CYP19 I.3/II promoter and thereby suppresses aromatase expression in preadipocytes. A, cells were treated with vehicle (control) or 10 μmol/L pioglitazone for 18 hours. Chromatin fragments were then immunoprecipitated with antibodies against pCREB, BRCA1, or p300 and the CYP19 I.3/II promoter was amplified by PCR (top) or real-time PCR (bottom). DNA sequencing was carried out, and the PCR product was confirmed to be the CYP19 1.3/II promoter. The CYP19 I.3/II promoter was not detected when normal IgG was used or antibody was omitted from immunoprecipitation step (data not shown). Mean ± SD are shown, n = 3. *, P < 0.01 versus vehicle-treated cells. B, cells were transfected with 0.9 μg CYP19 1.3/II promoter and 0.2 μg of pSVβgal. Cells also received 0.45 μg active CREB or 0.45 μg p300 expression vectors as indicated. The total amount of DNA received by cells in all treatment groups was maintained at 2 μg by using vector DNA. Subsequently, cells were treated with vehicle or 10 μmol/L pioglitazone for 24 hours. C, cells were transfected with 0.9 μg CYP19 1.3/II promoter and 0.2 μg of pSVβgal. Cells also received 0.9 μg of either control siRNA (GFP) or BRCA1 siRNA. Cells were then treated with vehicle or 10 μmol/L pioglitazone for 24 hours. Top, Northern blotting was conducted and the blot was probed for BRCA1 and 18S rRNA. B and C, luciferase activity was measured. Aromatase promoter activity represents data that have been normalized to β-galactosidase activity. B and C, mean ± SD are shown, n = 6. *, P < 0.01. D, cells were treated with vehicle (control) or 10 μmol/L pioglitazone for 24 hours. Cell lysates (500 μg) were subjected to immunoprecipitation with p300 antiserum and Western blotting was conducted for p300, pCREB, PPARγ, and BRCA1 as indicated. These proteins were not immunoprecipitated with control IgG. Input is p300. E, cells were treated with vehicle (control) or 10 μmol/L pioglitazone for 24 hours. Western blotting on cell lysate protein (100 μg/lane) was conducted and the immunoblot was probed as indicated.