Fig. 2

TC-HW-mediated cyclin D1 degradation dependent on GSK3β-mediated Thr-286 phosphorylation of cyclin D1. a HCT116 and SW480 were plated overnight and then treated with TC-HW (100 μg/ml) for the indicated times. Cell lysates were subjected to SDS-PAGE and the Western blot was performed using antibody against phospho-cyclin D1 (Thr-286). Actin was used as internal control for Western blot analysis. b HCT116 cells were transfected with wild type HA-tagged cyclin D1 or HA-tagged T286A cyclin D1 expression vector and then treated with 100 μg/ml of TC-HW. Cell lysates were subjected to SDS-PAGE and the Western blot was performed using antibody against HA-cyclin D1. Actin was used as internal control for Western blot analysis. *P < 0.05 compared to cell without TC-HW. c and d HCT116 and SW480 cells were pretreated with LiCl (20 mM), and then co-treated with TC-HW (100 μg/ml). Cell lysates were subjected to SDS-PAGE and the Western blot was performed using antibody against cyclin D1. Actin was used as internal control for Western blot analysis. *P < 0.05 compared to cell without TC-HW. e and f HCT116 and SW480 cells were pretreated with LiCl (20 mM), and then co-treated with TC-HW (100 μg/ml). Cell lysates were subjected to SDS-PAGE and the Western blot was performed using antibody against phospho-cyclin D1 (Thr-286). Actin was used as internal control for Western blot analysis. *P < 0.05 compared to cell without TC-HW