Fig. 5

Skp2 repression is required for NDRG2-driven post-translational regulation of p21 and p27. a p21 and p27 mRNA levels were determined in control or NDRG2-overexpressing HT29 and Caco-2 cells. b, c NDRG2 expression was upregulated after 2-day culture, which is correlated with the increased expression levels of p27 and p21, and decreased expression of Skp2. b Levels of indicated proteins in long-term confluence induced differentiation of Caco-2 and HT29 cells. The differentiation induction of Caco-2 and HT29 was performed as described under “Materials and methods” section. c Levels of indicated proteins under post-confluence growth induced differentiation. HT29 cells were seeded at 80% confluence at day 0 or continued to grow for an additional 1 or 2 days. d, e Skp2 protein d and mRNA e expression in HT29 cells with or without NDRG2 expression. f, g Levels of indicated proteins in HT29 cells with or without NDRG2 expression following 10 μg/ml CHX treatment for indicated time. p27 and p21 expression levels were further quantified by ImageJ and normalized to β-actin to determine the protein degradation rate. h, i NDRG2 attenuated p27 and p21 ubiquitination. HT29 cells were transfected with the indicated constructs together with pcDNA3.1( + )-3 HA-Ub and treated with 25 μmol/L MG-132 for 4 h before lysis. Anti-Flag immunoprecipitates from 1.5 mg aliquots of lysates were resolved by 7.5% SDS–PAGE and analyzed by western blotting with anti-HA antibody (top), or anti-FLAG antibody (middle). Cell lysates were subjected to western blotting with an anti-NDRG2 antibody (bottom). WCL whole-cell lysate