Figure 3.

Mouse p53 phosphomutants are not defective in inducing cell-cycle arrest. (A) Asynchronous p53^-/- MEFs were transfected with 5 µg of empty vector, wild-type p53 constructs, or phosphomutant p53 constructs, and were γ-irradiated at 20 Gy. Simultaneous staining of intracellular p53 (with Pab246 primary antibody and fluorescein isothiocyanate-conjugated anti-mouse IgG antibody) and genomic DNA (by propidium iodide) was performed before analysis by flow cytometry. p53-expressing cells, which emitted green fluorescence, were gated (except for vector-transfected controls), and their cell-cycle profile was determined by ModFIT cell-cycle analysis software (BD Biosciences). (B) Cell death was analyzed by determining sub-G₁ populations in cell-cycle analysis, as described in (A), by flow cytometry. Cells treated with and without 20 Gy γ-irradiation and harvested 24 hours after γ-irradiation were analyzed. The experiment was performed thrice independently. Data from three independent experiments are presented as mean ± SD. (C) p53^-/- MEFs were cotransfected with pcDNA-based constructs, and cells were selected with 2 µg/ml puromycin (Sigma) for 10 to 15 days and stained with crystal violet (Sigma). Colony formation assay was performed four independent times, and representative data are shown.