Additional file 2.

Presence of wt and mutant p53 in the mitochondrial fractions of HCT116 cultures in dependence of treatment. (A) HCT116 cultures were mock-treated (m) or treated with 10 μM α-amanitin (A), 375 μM 5FU (F), or α-amanitin plus 5FU (FA) for 24 h; the cells were fractionated and the quality of the fractionation was tested as described in Materials and methods. 15 μg of total protein (t) or mitochondrial protein (mt) were subjected to Western immunoblot analysis with either anti-p53 antibody DO-1 (1:2000) or anti-cytochrome oxidase IV (OX IV) antibody (1:1000). (B) HCT116 p53-/- cells were bulk-infected with retroviruses at a multiplicity of infection of <1 pfu/cell to express either p53 full-length mutant 175H or 273H from single gene copies per cell. The cells were mock-treated (-) or treated with 375 μM 5FU for 24 h (+), and where then fractionated and analyzed by immunoblotting (15 μg protein). total = total cell extract; cyto = cytoplasmic; mito = mitochondrial fraction. Again, proteins were detected with anti-p53 and anti-cytochrome oxidase antibodies. (C) Immune electron microscopy detects mutant p53 at the mitochondria of HCT116 p53-/- cells retrovirally infected to express empty vector, 175H or 273H. Gold grains (10 nm; arrows) indicative of the binding of anti-p53 antibody DO-1 were more frequently detected at mitochondria (mt) in cells harboring mutant p53 than in cells with no p53. The diagram outlines the numbers of grains counted in a blinded study at 105 mitochondria from randomly chosen microscopic fields; P values were determined with the Chi-square test.

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Heyne et al. Molecular Cancer 2008 7:54   doi:10.1186/1476-4598-7-54