Genomic DNA of TET1-overexpressing cells contains a modified nucleotide within the dinucleotide CG. (A) Description of experimental design. (B to F) Genomic DNA was purified from HEK293 cells overexpressing TET1 and cleaved with [(B) and (C)] MspI, (D) HpaII, or [(E) and (F)] TaqαI. The fragments were end-labeled, digested to 5′ dNMPs, and resolved by TLC. A modified nucleotide (subsequently identified as hm-dCMP) is indicated by ?. Neither 5m-dCMP nor the modified nucleotide are observed when the DNA is digested with HpaII. [(C) and (F)] Quantification of the relative abundance of dCMP, 5m-dCMP, and the modified nucleotide. Data shown are mean ± SD of three independent transfections and are representative of at least three experiments.