Fig. 3

Nkx2.5 promoter CpG island demethylation. a Position of Nkx2.5 gene on chromosome 5, sequence of the promoter region indicated. b After bisulfite conversion, the 160-bp promoter region of Nkx2.5 was amplified and cloned for bisulfite sequencing for control (U) and differentiated cardiomyocytes (D); shaded regions indicate the CpG islands that are modified after cardiac differentiation. c CpG islands were evaluated as methylated (orange dot) and unmethylated (red dot) sites by bisulfite sequencing. d After bisulfite conversion, methylation-specific PCR was performed for Nkx2.5 using unmethylated and methylated primers in control (U) and differentiated cardiomyocytes (D) (**p < 0.01, n = 3, all experiments performed in triplicate) (Color figure online)