(a) Schematic map of the 801-bp CpG island (−373 to +418) around exon 1 of the PRTFDC1 gene. CpG sites are indicated by vertical ticks on the expanded axis. Exon 1 is indicated by an open box, and the transcription start site is marked at +1. The fragment (Fragments 1–3) examined in a promoter assay is indicated by heavy black lines. The regions examined in COBRA and bisulfite-sequencing (Regions 1 and 2) are indicated by horizontal open bars. For COBRA, restriction sites for MluI and TaqI are indicated respectively by black or gray downward arrowheads. (b) Promoter activity of the PRTFDC1 CpG island. pGL3 basic empty vectors (mock) or constructs containing Fragments 1, 2, or 3 around exon 1 of PRTFDC1 (Figure 3a) were transfected with an internal control vector (pRL-hTK) into NA and HSC-2 cells. Luciferase activities were normalized versus an internal control. The data presented are the means±s.d. of three separate experiments, each performed in triplicate. (c) Representative results of COBRA of PRTFDC1 Region 1 and Region 2 in a panel of oral squamous-cell carcinoma cell lines after digestion with MluI and TaqI, respectively. Arrows show fragments specifically restricted in the sites recognized as methylated CpGs. An arrowhead indicates the unrestricted (unmethylated) fragment. As indicated by asterisks (*), eight cell lines were positive for restricted DNA fragments. (d) Results of bisulfite sequencing of the PRTFDC1 Region 1 (62 CpG sites) and Region 2 (33 CpG sites), examined in PRTFDC1-expressing and -nonexpressing cell lines. Each square indicates a CpG site: open squares, unmethylated; solid squares, methylated. Percent of solid squares in all squares analysed in each cell line was indicated by %methylation. NT, not tested.