Methylation status of the EphA5 gene promoter in prostate tissue. A, EphA5 methylation status was determined by MSP-PCR. All of the prostate cancer tissues exhibit complete methylation of the EphA5 gene. The unmethylated alleles were detected in adjacent noncancerous (top) and BPH (bottom) tissues. Lanes labeled “M” and “U” denote products amplified by primers recognizing methylated and unmethylated sequences, respectively. B, Methylation patterns of individual EphA5 promoter clones from prostate tissue that were bisulfite sequenced are shown. 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). Sample names and the methylation percentage of the corresponding tissue are indicated on the left and right sides, respectively. C, Representative examples of an unmethylated EphA5 CpG island in sample N1 (top) and a highly methylated CpG island in sample T24 are shown, as determined by bisulfite sequencing analysis. Arrows indicate positions of CpG dinucleotides.