Loss of methylation and induction of expression of pd-DMR genes under 5-aza-2′-deoxycytidine treatment. The cell line KMS11 was treated with demethylating low doses (200 nM) of DAC for 4 consecutive days and cell material was harvested at baseline, after DAC treatment, and after additional 17 days of culture after removal of DAC. Gene expression of pd-DMR genes was assayed by RT-PCR and products were run on a 2% agarose gel with glyceraldehyde-3-phosphate dehydrogenase as a housekeeping gene control (A). DNA methylation at the genomic loci of the pd-DMR probes was assayed by bisulfite pyrosequencing for the different time points. Black areas in the pie charts represent % methylated, white area % unmethylated cytosine residues. Pyrosequencing assay design for the probe mapping to GPX3 was technically impossible due to very high CpG density at the surrounding region of the pd-DMR array probe (B).