Fig. 5

Cyclin D1 degradation by DKC-E70 is followed by T286 phosphorylation dependent on ERK1/2, p38 and GSK3β. a HCT116 cells were treated with DKC-E70 (50 μg/ml) for the indicated times. b HCT116 cells were transfected with wild type HA-tagged cyclin D1 or HA-tagged T286A cyclin D1 expression vector for 24 h, and then treated with DKC-E70 (50 μg/ml) for 10 h. c HCT116 cells were pretreated with PD98059 (40 μM) as an ERK1/2 inhibitor, SB203580 (40 μM) as a p38 inhibitor or LiCl (20 mM) as a GSK3β inhibitor, and then co-treated with DKC-E70 (50 μg/ml) for 3 h. Cell lysates were subjected to SDS-PAGE and the Western blot was performed using antibodies against p-cyclin D1 (T286) or HA-cyclin D1. Actin was used as internal control for Western blot analysis. *P < 0.05 compared to cell without DKC-E70 treatment. d HCT116 cells were transfected with wild type HA-tagged cyclin D1 or HA-tagged T286A cyclin D1 expression vector for 24 h, and then treated with DKC-E70 (50 μg/ml) for 24 h. Cell viability was measured using MTT assay. *P < 0.05 compared to cell without DKC-E70