FIG. 7.

Phospho-mimicking mutation at Thr18 increases p53 activity. (A) H1299 cells were transfected with WT p53 or the T18A or T18D mutant and a luciferase reporter driven by a p53 response element derived from the p21 promoter. Luciferase activity was determined 16 to 24 h later. (B and C) The Tet-on U2OS-shp53 cells that inducibly express p53 shRNA were grown in the presence of doxycycline and transfected with HA-tagged, RNA interference-resistant WT p53 or a mutant and a luciferase reporter driven by either the p21 promoter (B) or a p53 response element derived from the Lats2 promoter (C). The luciferase activity was then assayed as for panel A. Results from three independent, duplicated experiments are presented. (D and E) Expression of the endogenous p21 gene was elevated by transfection with the p53 T18D mutant. RNA was isolated from transfected H1299 (D) or Tet-on U2OS (E) cells and processed for RT-PCR. (F) Western blots showing relative expression levels of WT and mutant p53 in transfected H1299 (upper panel) or Tet-on U2OS (lower panel) cells.