FIGURE 7.

JNK1/2 phosphorylation is involved in S1P-induced COX-2 expression. (A) After pretreatment with SP600125 for 1 h, cells were challenged with 15 μM S1P for 8 h. (B) Cells were transfected without or with COX-2 promoter-luciferase reporter gene, pretreated without or with SP600125 (10 μM) for 1 h, and then incubated with 15 μM S1P for 4 h (mRNA) or 1 h (promoter). The levels of COX-2 protein, mRNA expression, and promoter activity were determined by Western blot, real time-PCR, and promoter assay, respectively. (C) Cells were transfected with siRNA of JNK1 and then exposed to S1P for 8 h. (D) Cells were incubated with S1P (15 μM) for the indicated time intervals in the absence or presence of SP600125 (10 μM), W123 (10 μM), CAY (10 μM), GPA2A (10 μM), PTX (100 ng/ml), or Gö6976 (10 μM) for 1 h. The cell lysates were collected and analyzed by Western blot. The fold of basal was defined as normalization of the data to the respective “0,” and then compared the data of corresponding time points of control vs inhibitor with a statistic method, as described in the section of Methods. Data are expressed as mean ± SEM of three individual experiments (n = 3). ^# p < 0.05, as compared with the cells treated with S1P alone.