Figure 4

Role of MDM2 in GR inhibition by p53 and p73. A, Saos-2 cells were transfected with GR-luc DNA (200 ng) and increasing amounts of DNA encoding p53 (100, 250, 500, 750 ng). Where indicated cells were also transfected with DNA encoding siRNA directed against MDM2 (si MDM2). 24 hrs after transfection, cells were treated with dexamethasone (100 nM) for an additional 20 hrs, and luciferase activity in transfected cell lysates determined. Fold change in GR-luc activity is plotted (+/- s.e.m.) from multiple experiments compared to the activity of GR-luc alone, whose value is considered 1.0. GR, MDM2, and p53 immunoblots are shown below. B, MDM2/p53 double knockout (DKO) cells were transfected with GR-luc DNA (200 ng) and increasing amounts of DNA encoding p53 or p73 (50,100, 250, 500, 750, 1000 ng). Cells were treated with dexamethasone (100 nM) as described above, and luciferase activity in transfected cell lysates determined. Fold change in GR-luc activity is plotted compared to the activity of GR-luc alone, whose value is considered 1.0. Representative GR, p53 and p73 immunoblots are shown below. C, Saos-2 cells were transfected with GR-luc DNA (200 ng) and increasing amounts DNA encoding p73 (250, 500, 1000 ng). Where indicated cells were also transfected with the indicated amount DNA encoding MDM2 (250, 500, 1000 ng). Cells were treated with dexamethasone (100 nM) as described above and luciferase activity determined. Fold change in GR-luc activity is plotted. Representative GR, MDM2, and p73 immunoblots are shown below.