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Fig. 5 Alcohol causes p53/MDM2-dependent apoptosis in the mouse CNC line O9-1. (A) Immunostain reveals that p53 protein (red) is detected predominantly in the cytosol of untreated O9-1 cells, and this protein becomes stabilized within the nuclei (blue) 6hr after alcohol exposure. Right panel, alcohol increases the percentage of p53+ nuclei. (B) Alcohol exposure causes a six-fold rise in p53 protein content in O9-1 cells at 6hr following exposure. (C) Transfection of dominant-negative p53 (p53-DN) into O9-1 cells prevents the alcohol-induced apoptosis as detected using TUNEL (red signal) and does not affect the survival of unexposed cells, whereas transfection with the parent pCS2 vector does not affect cell survival. Right panel, alcohol increases the percentage of TUNEL+ cells and this is prevented by overexpression of dominant-negative p53 (+p53DN) but not by transfection with the empty vector (-p53DN). (D) Overexpression of MDM2 prevents the alcohol-induced apoptosis as detected by TUNEL (red) but does not affect the survival of control cells, compared with cells transfected with the empty vector pCS2. Right panel, alcohol increases the percentage of TUNEL+ O9-1 cells and this is normalized by MDM2. All values are mean ± SEM of 2 independent experiments as detailed in Methods. Data were analyzed using equal variance t-test (A, B) or one-way analysis of variance with Holm-Sidak multiple comparisons (C, D).