Steady-state level and transactivation ability of mouse p53 phosphomutants. (A) The alignment of the C-termini of human (accession no. NP_000537) and mouse (accession no. NP_035770) p53 proteins was performed by the Web-based alignment program BLAST2 (National Center for Biotechnology Information). Conserved residues are shown in the middle row between human and mouse p53 sequences. Serine residues at positions 375 and 378 for human p53, and at positions 373 and 375 for mouse p53, are indicated by boxes. (B) The S373 and S375 residues of mouse p53 were mutated to nonphosphorylatable alanine (A) residues or aspartic acid (D) that mimics phosphorylated residues, and were expressed in pCDNA or pBabe vectors. p53^-/- MEFs were cotransfected with 1 µg of empty vector, wild-type or mutant mouse p53, and 0.2 µg of GFP-encoding plasmids, and the steady-state level of p53 phosphomutants was determined by Western blot analysis. (C) The transcriptional activity of the mouse phosphomutants was determined by luciferase assays, using mdm2 promoter-luciferase construct. Luciferase activity was determined by chemiluminescence and was normalized against β-galactosidase activity. The experiment was performed thrice independently. Data from one of the experiments, performed in duplicate, are presented as mean ± SD. (D) Relative luciferase activity using the mdm2 promoter-luciferase construct was determined in mouse p53-transfected γ-irradiated (10 Gy) p53^-/- MEFs, as described above. The experiment was performed twice independently. Data from one of the experiments, performed in duplicate, are presented as mean ± SD. (E and F) Relative luciferase activity using p21 (E) and mdm2 promoter-luciferase constructs (F) together with human wild-type or phosphomutant IND constructs in p53-null H1299 cells. Data from one of the experiments, performed in duplicate, are presented as mean ± SD. (G and H) The expression of endogenous p53 target genes (bax, noxa, mdm2, and p21) in vector-transfected and p53-transfected p53^-/- MEFs (G) or human H1299 cells (H) was determined by semiquantitative RT-PCR and normalized against endogenous gapdh RNA level as described previously.

Mouse p53 phosphomutants, unlike their human counterparts, respond to ER stress as efficiently as wild-type p53. (A) p53^-/- MEFs were transfected with empty vector, wild-type mouse p53, or phosphomutant mouse p53, and then treated with vehicle (DMSO), 10 µg/ml TM, or 1 µM TG for 8 hours to induce ER stress. Cells were fixed and stained with anti-p53 antibody and propidium iodide before fluorescence confocal microscopy. Representative images, in which green fluorescence represents p53 protein and red fluorescence represents genomic DNA, are shown. Arrows indicate cells in which p53 is cytoplasmic. (B) Quantitative analysis of cells having cytoplasmic p53. Approximately 500 p53-expressing cells were scored and classified into cells with or without nuclear p53 stain. Data from one of the experiments, performed in triplicate, are presented as mean ± SD. (C) Similar experiments were carried out as described in (A) and (B) and were analyzed using another anti-p53 antibody (Pab1620). (D) H1299 cells were transfected with empty vector, wild-type human p53, or phosphomutant human p53 (376A or 376-378A[3A]). ER stress was induced by 1 µM TG, as described in (A). Approximately 500 p53-expressing cells were scored and classified into cells with or without nuclear p53 stain. Data from one of the experiments, performed in triplicate, are presented as mean ± SD. All experiments were performed at least thrice independently.

Mouse p53 does not bind 14-3-3. (A) Empty pcDNA vector and wild-type human or mouse p53 were transfected into H1299 or p53^-/- MEFs, respectively. Whole-cell lysates were immunoprecipitated with anti-14-3-3 antibody and analyzed by Western blot analysis using a mixture of Pab240 and DO-1 antibodies (left panel) or using these antibodies separately (right panel; Pab240 for mouse p53, and Pab421 for human p53) to detect p53 and K19 antibodies, which, in turn, detect 14-3-3. (B) Human MCF-7 and primary MEFs were γ-irradiated and collected after 3 hours, and 14-3-3 proteins were immunoprecipitated for Western blot analysis as described in (A), using Pab421 and Pab240 to detect p53. (C) Human diploid fibroblast cells IMR90 and MRC5 were γ-irradiated with 10 Gy, and the association of p53 with 14-3-3 was analyzed as described in (A), using DO-1 to detect human p53. All immunoprecipitation experiments have been repeated three times or more. (D) Western blot analysis of the phosphorylation status of mouse p53. Wild-type primary MEFs cells were γ-irradiated with 10 Gy and harvested at various time points as indicated. The phosphorylation state of p53 was analyzed with Pab421 antibody, the total amount of p53 was determined by CM5 antibody, and actin was used as loading control. Right panel: Blot with equal p53 protein loaded.

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.