Reactivation of silenced TSGs in colon cancer cells by SGI-1027. RKO cells were treated with varying concentrations of decitabine (Deci) and SGI-1027 continuously for different periods. A, real-time RT-PCR analysis of TIMP3 in RNA from cells treated with the inhibitors for 5 d. Normalization was done using β-actin as reference gene. Mean of three independent experiments. B, RT-PCR analysis of P16 and MLH1 in cells treated with the inhibitors for 7 d. C, quantitative analysis of P16 and MLH1 mRNA level in cells treated with decitabine or SGI-1027. Mean of three independent experiments. D, cells were treated with varying concentrations of decitabine and SGI-1027 continuously for 12 d. Whole-cell extracts were prepared and separated on a SDS-polyacrylamide gel and immunoblotted against antibodies specific for human DNMT1, TIMP3, P16, and Ku-70.