Fig. 7. Hint1 represses endogenous Cyclin D1 expression in SW480 cells. (A) Total RNA was isolated from SW480 cells transiently transfected with Hint1 expression plasmid or pCS2+ as a control. One-step RT-PCR was performed with total RNA and PCR products were analysed after the indicated number of cycles by agarose gel electophoresis. ß-Actin was used as a loading control. The presented experiment is representative of four independent transfections. (B) The amount of PCR products was quantified on a FujiFilm LAS-1000 system. The average of four independent RT-PCR experiments with total RNA from independent transfections is presented. (C) Western blot analysis of Cyclin D1 in cell lysates of SW480 cells transiently transfected with Hint1 expression plasmid. -Actinin was used as a loading control. (D) Dose-dependent repression of Cyclin D1 promoter in reporter gene assays with cyclin D1-luciferase constructs containing wild-type promoter or promoter with mutated TCF binding-sites.