Fig. 2

MLN4924 causes dose- and time-dependent accumulation of TOP1 and blocks TOP1 ubiquitination. A AMC-HN-8 and CAL-27 cells were treated with different doses of 10-HCPT (0, 10, 20, 40, and 80 µM) for 24 h, and then subjected to western blotting using antibodies against TOP1, p-CHK1 (S345), and t-CHK1. Actin was used as a loading control. B AMC-HN-8 and CAL-27 cells were treated with 20 µM 10-HCPT for 0, 0.5, 1, 2, and 4 h. C MLN4924 increased TOP1 levels in a dose-dependent manner. Cells were treated with various concentrations of MLN4924 (0, 0.25, 0.5, and 1 µM) for 24 h, and then western blotting was performed using TOP1, CUL1, p21, cyclin B1, and β-catenin antibodies. D MLN4924 extended the half-life of TOP1 following 10-HCPT treatment. Cells were treated with CHX (100 μg/ml) or 10-HCPT (40 μM), alone or in combination with MLN4924 (1 μM) for the indicated time periods, and then incubated with the indicated antibodies. E MLN4924 treatment attenuated TOP1 polyubiquitination induced by 10-HCPT treatment. HEK293 cells were transfected with the indicated plasmids for 48 h and then treated with 10-HCPT (40 μM) and MG132 (20 μM) alone or in combination with MLN4924 (1 μM) for 5 h. Immunoprecipitation with anti-HA beads was followed by IB with the indicated antibodies. WCE, whole-cell extract