Representative results of the methylation analyses of the NKX3.1 promoter. (A) Methylation-specific PCR. A visible PCR product in Lanes U indicates the presence of unmethylated alleles whereas a PCR product in Lanes M indicates the presence of methylated alleles. The left panel illustrates the methylation status of selected TGCTs and the testicular cancer cell lines. Note the methylation of sample # 2110. The right panel shows the unmethylated status of primary prostate cancers and prostate cancer cell lines. Abbreviations: NB, normal blood (positive control for unmethylated samples); IVD, in vitro methylated DNA (positive control for methylated samples); neg, negative control (containing water as template); U, lane for unmethylated MSP product; M, lane for methylated MSP product. (B) Bisulphite sequencing. The bisulphite sequence allows a positive display of 5-methyl cytosines in the gene promoter as unmethylated cytosines appear as thymines, while 5-methylcytosines appear as cytosines in the final sequence. The left chromatogram represents a part of the unmethylated NKX3.1 promoter in the germ cell tumor cell line TERA2, including 11 CpG sites marked by underlined letters. The right chromatogram represents the unmethylated prostate cancer cell line DU145. Both sequences have been generated by reversing the respective anti-sense sequences by use of the software "Chromas".