Fig. 5

FECR1 induces DNA demethylation in the FLI1 promote. a CpG islands in the FLI1 promoter. In order to detect the status of DNA methylation in FECR1-expressing cells, we designed five pairs of methylation-specific primers located at each CpG island. Three restriction enzymes were used to separate methylated and unmethylated DNAs. b FECR1 induces DNA demethylation. DNA methylation was measured by combined bisulfite restriction analysis (COBRA). PCR products from FECR1-expressing cells and vector control cells were digested by TaiI, Bsh1236I, and BstB1 to separate the unmethylated and methylated DNAs. They recognize and digest the methylated ACGT, CGCG, and TTCGAA sites, respectively. After treatment with sodium bisulfate, unmethylated cytosines were converted to uracils, and the ATGT, TGTG, and TTTGAA sites are not digested by these enzymes. After digestion, unmethylated and methylated DNAs were separated on 3% agarose gels. Note the uncut demethylated bands in FECR1-expressing cells