Figure 6. JNK signaling pathway regulates PES1 promoter activity and expression.

(A) pGLB-PES11 (−274/+172) was co-transfected with c-Jun, c-Jun-S63A/S73A and control vector into HCT116 cells. After 48 hr of transfection, the cells were treated with the following inhibitors for another 36 hr, including U0126 (10 µM), LY294002 (10 µM), and SP600125 (10 µM). DMSO was used as control. Luciferase activity was normalized to Renilla luciferase activity.The luciferase of cells transfected with control vector, which was treated with DMSO, was set to 1. (B) Cells were treated with inhibitors as in (A) and Western blot analysis was performed to check PES1 expression regulated by these inhibitors in HCT116 cells. (C) Western blot analysis of PES1 expression affected by varying concentration of SP600125 (0–50 µM). Total and phosphorylated c-Jun were also examined. GAPDH was used as a loading control. (D) pGLB-PES1 (−274/+172) was co-transfected with siRNA-JNK or siRNA-control. After 72 hr of transfection, luciferase activity was detected and was normalized to Renilla luciferase activity. Expression of JNK1 and PES1 proteins were determined by Western blot. Expression of PES1 mRNA was detected by quantitative RT-PCR.