FIG. 3.

Upregulation of p53 by Mdm2 siRNA induces expression of p21 protein and mRNA to a level comparable to that seen with treatment with doxorubicin. (A) Clones of U2OS cells stably transfected with control (cont.) or p53 shRNA were transiently transfected with control or Mdm2 siRNA or treated with 0.1 μg/ml doxorubicin as indicated. Cell lysates were immunoblotted (left panel), and corresponding dishes were subjected to RT-PCR analysis (right panel). (B) HCT116 and U2OS cells were cotransfected with 200 ng of a luciferase reporter construct containing the p21 5′ response element, control or Mdm2 siRNA, and 5 ng of pRL-Renilla for internal control. At 24 h after transfection, cells were treated with 0.5 μg/ml doxorubicin when indicated. At 48 h after transfection, cells were lysed and assayed for luciferase and Renilla activities. The indicated values represent the averages of data obtained in three independent experiments, each performed in duplicate. Error bars represent standard errors of the means.