Figure 3

High glucose (HG) culture condition does not affect drug-induced p53 stability and nuclear localization, rather it inhibits p53Ser46 phosphorylation. (A) HCT116 and A549 cells were treated with ADR (2 μg/ml) or CDDP (5 μg/ml), respectively, with or without HG culture condition. Equal amount of nuclear and cytoplasmic extracts was analysed by western immunoblotting with specific anti-p53 antibody. Anti-lamin A and anti-tubulin antibodies were used as control of efficient nuclear and cytoplasmic extraction, respectively. (B) HCT116, 2008 and A549 cells were treated with ADR or CDDP, as shown, with or without HG. Equal amount of total cell extracts was analysed by western immunboltting with phospho-specific Ser46 and Ser15 antibodies and total p53 antibody. Anti-β-actin was used as protein loading control. (C) H1299 cells were transiently co-transfected with p53AIP1-luc reporter and wtp53 or Ser46D mutant plasmids. Twenty-four hours after transfection culture medium was changed with a medium containing 2% FBS with or without HG. Results, normalized to β-gal activity are the mean ±S.D. of three independent experiments performed in duplicate. *P=0.001; NSS: not statistically significant.