Figure 3

ET-1-induced COX-2 expression is mediated through PI3K/Akt cascade. (A,B) Cells were pretreated with various concentrations of LY294002 (3, 30, or 300 nM) or SH-5 (1, 10, or 100 nM) for 1 h, and incubated with 10 nM ET-1 for 6 h (A) or 1 h (B). (C) Time dependence of ET-1-induced Akt phosphorylation; cells were treated with 10 nM ET-1 for the indicated time intervals in the presence or absence of SH-5 (10 nM). The phosphorylation of Akt was analyzed by Western blot using an anti-phospho-Akt antibody. (D) Cells were transfected with p85 (sh-p85) or Akt (sh-Akt) shRNA for 24 h (Topo: as a control) and incubated with 10 nM ET-1 for 6 h. The COX-2 protein (A,D) and mRNA (B) were analyzed by Western blotting and RT-PCR, respectively. Data are expressed as mean ± SEM of three individual experiments (n = 3). ^* P < 0.05, ^# P < 0.01 as compared with cells stimulated by ET-1 alone.