Figure 4

AP-1/c-Jun is essential for ET-1-induced COX-2 expression. (A,B) Cells were pretreated with various concentrations of tanshinone IIA (TSIIA, 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 c-Jun/AP-1 phosphorylation; cells were treated with 10 nM ET-1 for the indicated time intervals in the presence or absence of TSIIA (100 nM). The phosphorylation of c-Jun/AP-1 was analyzed by Western blot using an anti-phospho-c-Jun antibody. (D) Cells were transfected with siRNA of c-Jun (si-c-Jun) or scrambled (scrm; as a control) for 24 h 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. (E) Involvement of c-Jun/AP-1 in PGE₂ release induced by ET-1; cells were pretreated with TSIIA (100 nM) or transfected with c-Jun siRNA (si-c-Jun) and then incubated with ET-1 (10 nM) for 6 h. The conditioned media were collected to assay PGE₂ level by EIA kit. Data are expressed as mean ± SEM of three individual experiments (n = 3). ^# P < 0.01 as compared with cells stimulated by ET-1 alone.