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. 2015 Oct 23;172(22):5360–5376. doi: 10.1111/bph.13326

Figure 2.

Figure 2

S1P induces COX‐2 expression via PI3K/Akt‐dependent mTOR. (A) HTSMCs were pretreated with LY294002 (LY), SH‐5 or rapamycin (Rapa) for 1 h, and then incubated with S1P for 6 h. The COX‐2 protein expression was determined by Western blot. (B) Cells were pretreated with LY294002 (LY, 10 μM), SH‐5 (100 nM) or Rapamycin (Rapa, 1 μM) for 1 h, and then incubated with S1P for 4 h or 1 h. The COX‐2 mRNA expression and promoter activity were determined by real‐time PCR and promoter assay, respectively. (C) Cells were pretreated without or with W123, CAY10444, LY294002, SH‐5 or rapamycin for 1 h or edaravone, apocynin or DPI for 2 h, and then incubated with S1P (30 μM) for the indicated time intervals. The levels of phospho‐mTOR and phospho‐Akt were determined by Western blot. (D) Cells were transfected with siRNA of scrambled, p110, mTOR or Akt, and then incubated with S1P for 6 h. The protein levels of p110, mTOR, Akt and COX‐2 were determined by Western blot. Data are expressed as mean (A, C and D) or mean ± SEM (B) of five independent experiments. *P < 0.05; # P < 0.01, as compared with the cells exposed to S1P alone or S1P + scrambled siRNA (D).