Figure 2.

Translational repression of p53 by RoR. (A) Effect of RoR-siRNA on the p53 level with or without doxo. MCF-7 cells were first transfected with control or RoR-siRNA-2 overnight and then treated with doxo at the indicated concentrations for 16 h before harvesting for western blot. (B) RoR-siRNA induces p53 without causing p53 phosphorylation or acetylation. For doxo treatment, MCF-7 cells were transfected with control siRNA or RoR-siRNA-2 overnight and then treated with doxo at 0.5 μg/ml for 24 h. (C) Effect of RoR-siRNA combined with nutlin on p53. MCF-7 cells were transfected with control siRNA or RoR-siRNA-2 and then treated with nutlin at 0.5 μg/ml for 24 h before the cells were harvested for western blot. (D-F) RoR-siRNA does not change the stability of the doxo-induced p53. MCF-7 cells were transfected with control siRNA or RoR-siRNA-2 and then treated with CHX at 20 μg/ml for 30 min, followed by doxo treatment at 0.5 μg/ml. The cells were harvested for western blot at the indicated time points after doxo treatment. (G) RoR-siRNA increases the newly synthesized p53 level as detected by pulse-chase experiments. MCF-7 cells were first transfected with control siRNA or RoR-siRNA-2 overnight and newly synthesized protein was labeled with ³⁵S methionine, followed by IP with p53 antibody.