Methylation analysis of PF4 gene in primary MM, MM cell lines, and normal cells. (A) Schematic diagram showing the location of CpG sites at the 5′ promoter region and exon 1 of PF4 gene. Black arrows show the primers used for the amplification of the PCR product for bisulfite sequencing. Relative positions of CpG dinucleotides are indicated by vertical lines. The graphic below illustrates the results of bisulfite sequencing, with filled boxes indicating methylation and white boxes unmethylation at each CpG site. Five clones are analyzed for each sample. (B) 5′-Aza-2′-deoxycytidine (5′-Aza) demethylation treatment in MM cell lines. Restoration of PF4 expression in all MM cell lines was observed after treatment with 1, 3, 5 or 10 μM of 5′-Aza for 4 days. ACTB expression was examined for internal control.