Figure 3.

DNA methylation is absent at the region −73 to +158 region of GM2-synthase gene in normal and RCC cell lines. A, Region P is located within a CpG island. Schematic representation of predicted CpG island using MethPrimer database around the first TSS of GM2-synthase gene encompassing Region P. B, DNA methylation is absent at the −73 to +158 region confirmed by BstUI digestion assay. Isolated genomic DNA from indicated cell lines were treated with bisulphite (left gel) or untreated (right gel). Following PCR, DNA samples were digested with BstUI restriction enzyme. Restriction sites of BstUI within the DNA are indicated by arrowheads (not to scale). The primers used to amplify genomic regions of GM2-synthase gene are listed in Table S1H. C, DNA methylation is absent at the region −73 to +158 region confirmed by bisulphite sequencing. Methylation status of 17 separate TA clones of NKE, 15 and 9 separate TA clones of SK-RC-45 and SK-RC-26B, respectively, were determined by bisulphite sequencing analysis. The filled or open circles represent the methylated or unmethylated CpG sites, respectively, and each horizontal column designates the arbitrary position of a CpG dinucleotide around the TSS of GM2-synthase gene.