RG108 (N-Phthalyl-L-tryptophan) is a DNA methyltransferase inhibitor with an IC50 of 115 nM.

DNA methyltransferase:115 nM

In iPS cells derived from SM, RG108 significantly reduces DNA activity. In tumor cells, RG108 inhibits gene demethylation and reactivation without affecting the methylation of telomeric gene sequences.

In vitro methylation assay: The substrate DNA for the in vitro methylation assay is a 798 bp fragment (?423/+375 relative to the initiation codon) from the promoter region of the human p16Ink4a gene. The methylation reaction contains 350 to 400 ng substrate DNA and 4 units of M.SssI methylase (0.5 μM) in a final volume of 50 μL. Inhibitors are added to final concentrations of 10, 100, 200, and 500 μM, respectively. Reactions are done at 37 °C for 2 hours. After completion, the reaction is inactivated at 65 °C for 15 minutes and the DNA is purified using PCR Purification kit. Three hundred nanograms of purified DNA is digested for 3 hours at 60 °C with 30 units of BstUI and analyzed on 2% Tris-borate EDTA agarose gels.

For the determination of cellular growth and viability, cells are stained with trypan blue and counted using a standard counting grid.(Only for Reference)