Figure 4

Dominant inhibitory effect of p53DD on transcription but not mitochondrial apoptosis. (A) Western immunoblot on 30 μg of total protein extract from HCT116 cells expressing vector-only or p53DD, documenting the expression levels of endogenous wt p53 and p53DD. p53DD was detected with antibody PAb421 (1:200); loading control β-actin was detected with anti-β-actin antibody diluted at 1:5000. (B) RT-PCR on total RNA prepared from the indicated cell lines at 24 h after mock-treatment or treatment with 375 μM 5FU. The primers were specific for the p53-regulated p21 transcript and for the gapdh transcript as a control. (C) Percentage of apoptosis in the indicated cell lines and at 48 h after mock-treatment or exposure to α-amanitin (10 μM; A), 5FU (375 μM; F), or both combined (FA). Apoptosis was measured by determining the numbers of cells with a sub-2n DNA content after PI staining, as described in Materials and methods.