Figure 3
The MEK/ERK inhibitor PD0325901 is responsible for downregulation of UHRF1 and DNMT1 expression at the level of transcription in HCT116 cells. (a,b) HCT116 cells were treated without or with PD0325901 or CHIR99021 individually or in combination for 2 days before harvested for analysis of the levels of proteins (a) and mRNAs (b). (c) Western blotting analysis showing that PD0325901 treatment completely blocked ERK1/2 phosphorylation in HCT116 cells. The cells were treated with or without PD0325901 for 12 hours. (d–f) Time course experiments showing the kinetics of 2i, CHIR99021 and PD0325901 treatment on the down-regulation of UHRF1 and DNMT1 proteins (d), UHRF1 mRNA (e) and DNMT1 mRNA (f). Note that PD0325901 had the same effect as 2i. (g,h) Luciferase reporter assays showing the response of the UHRF1 and DNMT1 proximal promoters to MEK1/2 inhibitor PD0325901. The control PGL3-luc and PGL3-luc containing either UHRF1 (g) or DNMT1 (h) proximal promoter were transfected into HCT116 cells and treated with or without 1 μM PD0325901 for 24 hours before harvested for standard luciferase assay. The effect of PD0325901 on ERK1/2 phosphorylation was confirmed by western blotting analysis.
