Fig. 6.

Transcriptional and translational regulation of p53 in HeLa cells. a HeLa cells were transiently transfected with 0.5 μg of pSG5.HA vector or the indicated amount of pSG5.HA.p53 and incubated for 12 h with 5 mM metformin. The cell lysates were subjected to western blotting with antibodies against p53, DEC1, and PARP. ACTN was the loading control. The protein levels of p53, DEC1, and cPARP after normalization with the loading control protein ACTN are presented as fold change. b HeLa cells were incubated for 5 h with the indicated concentrations of metformin with or without 10 μM MG132, after which the cell lysates were subjected to western blotting with an antibody against p53. ACTN was the loading control. The protein levels of p53 after normalization with the loading control protein ACTN are presented as fold change. c and d HeLa cells were incubated for 12 h with the indicated concentrations of metformin with and without 0.1 μM actinomycin D (Act D) or 50 μg/ml cycloheximide (CHX). Levels of p53 mRNA and protein were then assayed in the cell lysates using RT-PCR (c) and western blotting (d), respectively. GAPDH mRNA was the mRNA loading control; ACTN was the protein loading control. e and f HeLa cells were incubated with 5 mM metformin (e) or 50 μg/ml CHX (f) for the indicated times, after which cell lysates were subjected to western blotting with an antibody against p53. g HeLa cells were incubated for the indicated times with 10 mM metformin with and without 50 ng/ml CHX. The cell lysates were then subjected to western blotting with an antibody against p53. d-g The protein levels of p53 after normalization with the loading control protein ACTN are presented as fold change. The results are representative of three independent experiments