The distal PDPN promoter is fully methylated in MG63 but non-methylated in Saos-2 cells. A) Schematic representation of Hpa II/Msp I binding sites and CpG islands within a Pst I genomic DNA fragment spanning from bp -2799 to +1191 of the PDPN promoter with respect to the transcription initiation start site (arrow at +1). The graphs depict the CpG blot created by the EMBOSS program. Exon 1 is drawn as an open box in the sequence. Hpa II/Msp I recognition sites are represented by vertical lines along the sequence, and CpG islands are indicated as empty boxes above. Probes used for Southern blot analysis and stretches that were amplified after bisulfite genomic DNA sequencing are indicated as black bars and lines, respectively, below the sequence. B) Genomic DNA from MG63 and Saos-2 cells was double-digested with Pst I, and with Pst I in combination with the methylation-sensitive restriction enzyme Hpa II or the methylation-insensitive restriction enzyme Msp I. DNA then was fractionated by agarose gel electrophoresis followed by Southern blotting using the five DNA probes depicted in Fig. 8A. C) CpG methylation pattern of the PDPN promoter and of exonic and intronic CpG islands in MG63 cells as determined by sodium bisulfite genomic DNA sequencing. The CpG number indicates the order of the CpG site inside the Pst I genomic fragment, and the CpG position denotes its position in relation to the transcription start site. The color-tone of the circle reflects the degree of methylation: black: 100%, dark blue: 99-67% blue: 66-34%, light blue: 33-1%, white: 0% methylation. Asterisks indicate CpG motifs that are part of a Hpa II/Msp I restriction digest site.