Figure 4

High glucose (HG) induces calyculin A-sensitive phospahatase(s) able to target p-Ser46. (A) HCT116, cells were treated with ADR with or without HG; phosphatase inhibitor calyculin A was added at 1 nM along with ADR. Equal amount of total cell extracts was analysed by western immunboltting with phospho-specific Ser46 antibody and total p53 antibody. Anti-β-actin was used as protein loading control. (B) RNA samples were extracted by HCT116 cells treated as in (A) and used for RT-PCR of p53 target gene expression. 28S levels were analyzed as internal control (left panel). Densitometric analysis is shown in the right panel and plotted as the expression ratio to control 28S expression. (C) Tunel assay of HCT116 and 2008 cells treated with ADR and CDDP, respectively, with or without HG condition. Phosphatase inhibitor calyculin A was added at 1 nM along with drugs. The results shown are the percentage of Tunel positive cells and are representative of two independent experiments performed in duplicates, ±S.D. *P=0.001.