Figure 7. Sirt1 regulates COX2 expression in cultured RMICs.

(A) Wild-type or Sirt1-knockdown cultured RMICs were challenged with H₂O₂ (500 μM) for 0, 3, or 6 hours. Expression of Sirt1, COX2, and COX1 was examined by immunoblot. (B) Wild-type or Sirt1-knockdown RMICs were challenged with H₂O₂ (500 μM) for 0 or 6 hours. COX2 mRNA expression was assessed by qRT-PCR (n = 4, *P < 0.0001 versus wild-type cells with H₂O₂). (C) RMICs were treated with the Sirt1 activator SRT2183 (0, 5, 10, 20 μM) for 8 hours, and COX2 expression was examined by immunoblot (n = 4). (D) COX2 luciferase reporter activity was measured in wild-type or Sirt1-knockdown RMICs with H₂O₂ (250 μM) for 0 or 12 hours by using the Dual Luciferase assay kit (n = 12, *P < 0.0001 versus wild-type cells with H₂O₂). (E) ChIP assay was performed on nontreated or H₂O₂-treated (500 μM, 3 hours) RMICs using anti-Sirt1 antibody and PCR primers recognizing mouse COX2 5′ sequences upstream of the transcription start site [–712, –396] or [–4,166, –3,946]. C, control: immunoprecipitation with normal rabbit IgG. Data are representative of 3 independent experiments.