5.

Inhibition of PKCa-ERK1/2-FRA-1 cascade abolished the HS induced malignant effects. (A) BrdU incorporation was detected by immunofluorescent staining. The percentage of BrdU positive cells (BPI) in CT-26 cells were quantitatively measured. (B) Sphere-forming assay of in control and HS-treated CT-26 and SW620 cells in the presence or absence of Gö6976 (300 nM), U0126 (5 μM), Fra-1 siRNA or c-Myc siRNA. Quantification of the spheres by MTT assay. (C) Luciferase reporter assay of c-Myc gene in CT-26 and SW620 cells with or without AP-1 promotor dysfunctional mutation. (D) Matrigel pre-coated Transwell assays were performed to examine the invasion ability of CT-26 and SW620 cells 3 days after HS treatment, in the presence or absence of Gö6976 (300 nM), U0126 (5 μM), Fra-1 siRNA or c-Myc siRNA. (E) Western blot analysis of MMP-1 expression in control and HS-treated CT-26 cells in the presence or absence of inhibitors or siRNAs. The quantification value was labeled above the bands. (F) Luciferase reporter assay of MMP-1 gene with or without AP-1 promotor dysfuctional mutation. P values were determined by two-sided Student’s t or ANOVA test. *P< 0.05, **P< 0.01. All data were from three independent experiments.