Fig. 2.

DNA methylation is altered at CpG sites in the CA9 promoter in metastatic implants from mouse xenografts. The DNA methylation status at the -6 CpG site was analyzed with the Illumina HumanMethylation 450 BeadChip (A) and qMSP (B). The DNA methylation status was analyzed by bisulfite sequencing (C). The CA9 promoter is located between positions 35673651 and 35674055 in the human GRCh37/hg19 assembly, and contains eight CpG residues on chromosome 9. The eight CpGs are at positions -197, -74, -19, -6, +4, +13, +40, and +86 relative to the transcription start site. Each circle represents a CpG dinucleotide. The methylation status of each CpG site is indicated with a black (methylated) or white (unmethylated) circle. The percentage of methylation at each site is indicated in a pie graph on the bottom line. The black segment of the pie graph indicates the percentage of methylated CpGs, whereas the white segment represents the percentage of unmethylated CpGs (C). Triangles above the circles in C indicate the specific CpG site that was used for qMSP. Statistical analyses were performed by one-way ANOVA and subsequent Bonferroni tests (^*p<0.05). M, the percentage of methylated CpGs; U, the percentage of unmethylated CpGs; qMSP, quantitative methylation-specific PCR; PCR, polymerase chain reaction; CA9, carbonic anhydrase 9; ANOVA, analysis of variance.