Figure 3

Mitochondrial localization of p53 and apoptosis in HCT116 cells infected to express mutant p53. (A) Western blots on 15 μg of total cellular protein (t) or mitochondrial protein (mt) prepared from the indicated cell lines at 24 h after mock-treatment or exposure to α-amanitin (10 μM; A), 5FU (375 μM; F), or both combined (FA). p53 was detected with anti-p53 antibody DO-1 at a dilution of 1:2000. p53-ΔC: one of the indicated ΔC-forms of mutant p53. (B) Percentage of apoptosis measured by flow cytometry in cultures of the indicated cell lines. The cells were either mock-treated or drug-treated as specified under. Apoptosis at the 12 and 24 h time points (early apoptosis) was determined by measurement of Annexin V binding as detailed in Materials and methods. The apoptosis at 48 h (late apoptosis) was determined by measurement of the cells with a sub-2n DNA content. (C) Western blot on cytoplasm prepared from cultures of the indicated cell lines at 48 h after mock- or drug-treatment. Cytoplasmic accumulation of cytochrome c (cyto c) as a hallmark of apoptosis became apparent after staining of the blot with anti-cytochrome c antibody at a dilution of 1:1000. Vector: HCT116 cells infected with vector-only. 175ΔC: HCT116 cells infected with retroviral vector expressing mutant 175H with a deleted C-terminus. 273ΔC: HCT116 cells infected with retroviral vector expressing mutant 273H with a deleted C-terminus.