Figure 6

Bisulfite sequencing of the M3R CpG island before and after 5-aza-2′-deoxycytidine (5-Aza-CdR) treatment. (a) The M3R CpG island (GenBank no. NM_000740) was analyzed. This sequence spans from base pair +235 to +649 (relative to the start codon) and includes 14 CG pairs inside exon 8, a coding region. The start (ATG) and stop (TAG) codons in exon 8 are shown. The CG sites within this sequence are underlined and shown in bold. TSS indicates transcription start site. (b) Bisulfite sequencing of CpG island of M3R in A253 cells. DNA isolated from A253 before (the left panel) and after 5-Aza-CdR treatment (the right panel). They were treated with bisulfite, and the M3R CpG island was PCR amplified. The resultant PCR product was ligated into pCR2.1-TOPO using the TA cloning system. Five clones before and after 5-Aza-CdR treatment were picked and sequenced. (c) Bisulfite sequencing of CpG island of M3R in human cancer tissue (the left panel) and normal tissue (the right panel). (d) Bisulfite sequencing of CpG island of M3R in SGT cells before (the left panel) and after 5-Aza-CdR treatment (the right panel). Symbols: ○, unmethylated cytosines; •, methylated cytosines.