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. 2020 Oct 30;11:569802. doi: 10.3389/fphar.2020.569802

FIGURE 4.

FIGURE 4

PKCα mediates S1P-induced COX-2 expression. (A) Cells were pretreated with Gö6976 for 1 h and then incubated with 15 μM S1P for 8 h. (B) Cells were transfected without or with COX-2 promoter-luciferase reporter gene, pretreated without or with Gö6976 (1 μM) for 1 h, and then incubated with 15 μM S1P for 4 h (mRNA) or 1 h (promoter). Western blot, real time-PCR, and promoter assay were performed to determine the levels of COX-2 protein, mRNA expression, and promoter activity, respectively. (C) Prior to exposure with S1P for 8 h, cells were transfected with siRNA of scrambled or PKCα. (D) Cells were incubated with S1P (15 μM) for the indicated time intervals. (E) Cells were incubated with S1P (15 μM) for 5 min in the absence or presence of Gö6976 (1 μM), W123 (10 μM), CAY (10 μM), GPA2A (10 μM), or PTX (100 ng/ml) for 1 h. The membrane and cytosol fractions were prepared and analyzed by Western blot. Data are expressed as mean ± SEM of three individual experiments (n = 3). # p < 0.05, as compared with the cells treated with S1P alone.