Figure 3.

Overexpression of SIRT1 inhibits PGE₂-mediated induction of aromatase. Human breast ASC cell line was used except in D, where primary human breast ASCs were employed. A and B, cells were treated with 500 nm PGE₂ for 0–24 h as indicated. C–E, cells were either untransfected (Control) or transfected as indicated. Cells were then treated with vehicle or 500 nm PGE₂ for 24 h. A–D, cell lysates were subjected to Western blotting, and the blots were probed as indicated. E, aromatase mRNA levels were determined by quantitative PCR. F, cells were transfected with 0.9 μg of aromatase promoter PII-luciferase and 0.2 μg of psvβ-gal constructs. In addition, as indicated, cells received 0.9 μg of control expression vector or SIRT1 expression vector. Cells were treated with vehicle (control) or 500 nm PGE₂ for 24 h. Cells were harvested, and luciferase activity was measured. Luciferase activity was normalized to β-gal activity. G, cells were either untransfected or transfected with 0.9 μg of control vector or SIRT1 expression vector. Subsequently, cells were treated with vehicle or 500 nm PGE₂ for 3 h. ChIP assays were performed. Chromatin fragments were immunoprecipitated with antibodies against HIF-1α, and the aromatase promoter was amplified by real-time PCR. DNA sequencing was carried out, and the PCR products were confirmed to be the aromatase promoter. This promoter was not detected when normal IgG was used or when antibody was omitted from the immunoprecipitation step. In E–G, means ± S.D. (error bars) are shown (n = 6). *, p < 0.001.