KEAP1 and Nrf2 expression. (A) KEAP1 mRNA expression in 10 CRC cell lines was evaluated by real-time PCR. The expression level in Colo320DM cells was arbitrarily designated as 1. Columns, mean (n = 3); bars, standard deviation (SD). (B, upper) KEAP1 mRNA levels in HT29 cells (methylated) and Colo320DM cells (unmethylated) were analyzed by real-time PCR after treatment with 5-Aza-dC, TSA, and 5-Aza-dC + TSA. The expression level in HT29 cells was arbitrarily designated as 1. Columns, mean (n = 3); bars, SD. *P < 0.05. (B, lower) MSP analysis of in HT29 cells and Colo320DM treated with 5-Aza-dC + TSA. M, methylation-specific primer; U, non-methylation-specific primer. (C) Western blot analysis of Keap1 and Nrf2 in methylated and unmethylated colon cancer cells. Whole-cell extracts (W), cytosolic extracts (C), and nuclear extracts (N) were prepared from Colo320DM and HT29 cells. Extracts were stained with antibody to Keap1 or Nrf2A. β-Actin, histone H1, and GAPDH antibodies were used as loading controls for whole-cell, cytosolic, and nuclear fractions, respectively.