Figure 8.

ATF3 is important for PGE₂-mediated down-regulation of SIRT1 in human breast ASCs. A–G, a human breast ASC cell line was used. A, cells were pretreated with vehicle or the indicated concentrations of H89, a PKA inhibitor, for 2 h and then treated with vehicle, 500 nm PGE₂, or 500 nm PGE₂ plus the indicated concentrations of H89 for 24 h. B, cells were treated with the indicated concentrations of PGE₂ for 2 h. Cell lysates were subjected to Western blotting, and the blots were probed as indicated. C, cells were pretreated with vehicle or the indicated concentrations of H89 for 2 h and then treated with vehicle, 500 nm PGE₂, or 500 nm PGE₂ plus the indicated concentrations of H89 for 24 h. D, cells were transfected with 0.9 μg of full-length SIRT1 promoter and 0.2 μg of psv-β-gal constructs. In the column labeled Control siRNA, cells received 0.9 μg of control siRNA, and in the column labeled ATF3 siRNA, cells received 0.9 μg of ATF3 siRNA. 24 h later, cells were treated with vehicle (control) or PGE₂ for 24 h. Cells were harvested, and luciferase activity was measured. Luciferase activity was normalized to β-gal activity. Inset, Western blotting was performed after transfecting ASCs with control or ATF3 siRNA. E, the top panel represents different deletions of SIRT1 promoter used. Full-length promoter and deletion 1 (Del1) contain a CRE site, whereas deletion 2 (Del2) lacks a CRE site. Cells were transfected with 1.8 μg of each of three SIRT1 promoter-luciferase constructs and 0.2 μg of psv-β-gal for 24 h. Subsequently, cells were treated with vehicle (control) or 500 nm PGE₂ for 24 h. Cells were then harvested, and luciferase activity was measured. Luciferase activity was normalized to β-gal activity. F, ChIP assay was performed. Cells were treated with vehicle (control) or 500 nm PGE₂ for 3 h. Chromatin fragments were immunoprecipitated with antibodies against ATF3, and the SIRT1 promoter was amplified by real-time PCR. DNA sequencing was carried out, and the PCR products were confirmed to be the SIRT1 promoter. This promoter was not detected when normal IgG was used or when antibody was omitted from the immunoprecipitation step. G, cells were either untransfected or transfected as indicated with 2 μg of siRNA to GFP (control siRNA) or ATF3 siRNA. 48 h after transfection, cells were treated with vehicle (control) or 500 nm PGE₂ for 24 h. Cell lysates were subjected to Western blotting, and the blots were probed as indicated. D–F, means ± S.D. (error bars); n = 6. *, p < 0.001.