Reversal of β-gal inactivation by 5-aza-C. (A) Nine MMR-deficient clones (HCT116–1, HCT116–2, HCT116–3, HCT116–4, HCT116–5, HCT116–6, RKO-1, DLD1–1, and LoVo-1) and the MMR-proficient clone HT29–6 were treated with different concentrations (0, 1, 2, 4, or 5 μM) of 5-aza-C for 72 hr and stained with X-Gal. In 8 of 9 MMR-deficient clones, 5-aza-C significantly increased the fraction of cells expressing β-gal in a dose-dependent manner. The single MMR-deficient clone in which 5-aza-C had no effect was LoVo-1. LoVo-1 had a truncating mutation in the β-gal gene (see text). (B) To determine whether the reversal of inactivation of β-gal expression by 5-aza-C was permanent, the drug (5 μM) was removed from the medium, and β-gal expression was measured at subsequent times. The clones reverted to the same basal level of β-gal expression found before 5-aza-C treatment.