Analysis of the p53 pathway using RT-PCR and a p53-Luciferase reporter assay.A) Relative levels of mRNA expression for genes involved in the p53 pathway determined by RT-PCR. For each gene the expression level of passage 6 CEF cells was set to 1.0. The levels for other samples were then adjusted accordingly. A minimum of three independent RT-PCR experiments were analyzed using the NIH image software program and normalized using the expression levels of GAPDH. Differences ≤0.05 were considered significant. Different letters indicate significant differences. B) p53 and p21 WAF1 mRNA expression levels were measured by RT-PCR in passage 43 and 95 SC-2 cells as well as in passage 280 DF-1 cells ± the protein synthesis inhibitor CHX. C) p53 protein levels in primary passage 6 CEF and passage 95 SC-2. A minimum of three independent Western blot experiments were analyzed using the NIH image software program and normalized using the expression levels of actin. Differences ≤ 0.05 were considered significant. D) Altered biological activity of p53 in immortal DF-1 and SC-2 cells using the luciferase reporter gene assay. All cells were cotransfected with a control reporter plasmid (Con-Luc) or p53 reporter plasmid (p53-Luc) and pcDNA3.1-LacZ plasmid. The β-lactamase (LacZ) gene was amplified by PCR to normalize transfection efficiency and the GAPDH gene was amplified by RT-PCR to account for loading differences. Levels of luciferase activity were determined using the Luciferase Assay System (Promega) following the manufacturer's instructions. Luciferase activity in the cells transfected with the Con-Luc plasmid were set to 1.0. The normalized values shown are relative expression levels with s.d. Differences ≤ 0.05 were considered significant. Different letters indicate significant differences.
Christman et al. BMC Cell Biology 2006 7:27 doi:10.1186/1471-2121-7-27