Figure 4.

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.