Figure 3

Wip1 phosphatase attenuates p53 activity. Wip1 and/or Tax expression reduces p53 transactivation of a pG13Luc-reporter in HCT-116 cells in the absence (A) or in the presence (B) of exogenous p53 (0.8 μg). HCT-116 cells were transfected with 0.2 μg of Tax and/or 0.75 μg of Wip1 expression plasmid (*: 0.01≤p≤0.05; **: p<0.05; t-test). (C) Cell lysates from a representative experiment were subjected to immunoblotting using anti-p53, anti-Tax, anti-Flag and anti-α-tubulin as indicated. The lane numbers of the samples in each case corresponds to the lane numbers indicated in panels (A) and (B). (D) Analysis of cell endogenous p53 activity was conducted using the pG13-Luciferase plasmid in Wip1^−/− and Wip1^+/+ Mouse Embryonic Fibroblasts (MEF). Top panel shows PCR genotypic characterizations of two independent Wip1^+/+ (60, 63) and two independent Wip1^−/− (7, 12) MEFs; each was assayed twice in pG13Luc-reporter assays. Bottom graph shows the luciferase assays. All luciferase activities were normalized to a co-transfected β-galactosidase reporter. Statistical significance was determined using t-test (*: p=0.0076). (E) Analyses of cell endogenous p21 and (F) p53 mRNAs in 5 independent Wip1^+/+ (left) and 4 independent Wip1^−/− MEFs. Real-time RT-PCR analyses of p53 and p21 and GAPDH (internal standard) transcripts were performed in Wip1^−/− and Wip1^+/+ MEFs. There was no statistically significant difference in p53 mRNA levels, while p21 mRNA levels were significantly different between Wip1^+/+ and Wip1^−/− MEFs (*: p=0.0425; t-test).