Methylation status of the EphA5 gene promoter in prostate cell lines. A, EphA5 methylation status was determined by MSP-PCR analysis. All prostate cancer cell lines exhibited complete methylation of the EphA5 gene. Unmethylated EphA5 alleles were detected in RWPE-1 and PC-3 cell lines. Lanes labeled “M” and “U” denote products amplified with primers recognizing methylated and unmethylated sequences, respectively. B, Schematic depiction of the EphA5 promoter-associated CpG island, which spans the region from -103 to +303 with respect to the TSS (+1). The bisulfite sequencing PCR primers are shown in light blue and bold type. The MSP = PCR primers are highlighted in khaki, italicized, and underlined. There are 38 CpG sites in this region; the CpG sites are numbered in red and bold type. C, Methylation patterns of individual EphA5 promoter clones from prostate cell lines that were sequenced using bisulfite methods. Six clones from each sample were bisulfite sequenced to obtain a representative sampling of methylation patterns; CpG dinucleotides are represented by squares (■, methylated cytokines; □, unmethylated cytosines). Cell-line names and the percentage of methylation for the corresponding cell line are indicated on the left and right sides, respectively. D, Representative chromatograms of CpG sites 14 to 18 obtained from bisulfite sequencing of the EphA5 fragment. Arrows indicate positions of CpG dinucleotides.