Figure 2

Dominant inhibition of wt p53 by the mutants 175ΔC and 273ΔC. (A) RT-PCR on the three HCT116 cell lines infected to express empty vector, 175ΔC, or 273ΔC, after mock-treatment (-) or treatment with 375 mM 5FU (+) for 24 h. The expression levels of the p21Waf/Cip1, bak, and bax genes, and of the gapdh control gene, are documented. (B) HCT116 p53-/- cells were transiently transfected to express empty vector or wt p53 in combination with one of the indicated mutants. The Western blots on 15 μg of total protein extract prepared at 24 h after transfection were incubated with anti-p53 antibody DO-1 (1:2000) or anti-p21 antibody (1:1000). (C) Chromatin immunoprecipitation (ChIP) assay on the three indicated cell lines. Depicted are the relative quantities of p21 promoter DNA precipitable with anti-p53 antibody DO-1 at 24 h after treatment with 375 μM 5FU vs. mock treatment. INPUT shows that the total quantities of p21 promoter DNA in the cell samples were similar. (D) Colony formation assay on HCT116 cells infected with empty vector or vector expressing 175ΔC or 273ΔC. One thousand live cells were seeded onto 10 cm dishes and were mock-treated or received between 10 μM and 0.5 μM etoposide. After 10 days in culture, colonies were fixed and stained with crystal violet, as described in Materials and methods.