Suppression of DNA methyltransferases is sufficient to induce demethylation.

<p>(A) Representative Western blot indicates that DNMT1 protein level in H719 cells was significantly decreased in the DNMT1-deficient stable clone when compared to the non-specific oligonucleotide treated control. (B) and (C) Representative Western blots also show that DNMT3A or DNMT3B protein level was significantly decreased after RNAi treatment in DKD of DNMT1/DNMT3A cells or DKD of DNMT1/DNMT3B cells, respectively. β-actin is shown as a loading control in the lower panel. (D) A schematic diagram showing the <i>p21^Waf1/Cip1</i> promoter, with the black region indicating the fragment which underwent bisulfite sequencing (−233 to +2 relative to transcription start site). (E) With bisulfite sequencing, 10 clones were randomly selected and the methylation status of the <i>p21^Waf1/Cip1</i> promoter was determined in untreated H719 cells or in the H719 cells treated with knockdown of DNMT1, DKD of DNMT1/DNMT3A or DKD of DNMT1/DNMT3B. (F) RNA was extracted from H719 cells treated with DKD of DNMT1/DNMT3B, and RT-PCR was performed to detect expression of p21 mRNA. β-actin is shown as a loading control for the RT-PCR analysis.