Demethylation studies using K562 cells treated with 5-aza-2′-deoxycytidine.

Cells (10⁶) were treated with 1 μM 5-aza-2′-deoxycytidine for 24 hours and cells were then washed and fresh medium was added. The treated cells were harvested at days 1, 2, 3, 4, and 5 after 5-aza-2′-deoxycytidine treatment for JunB expression and methylation-specific PCR analysis. (A) JunB gene expression levels in K562 cells were increased (decreased ΔC_T) after 5-aza-2′-deoxycytidine treatment for 24 hours and persisted for 5 days as determined by real-time quantitative RT-PCR. Data are presented as mean ± SE calculated from 5 duplicate studies. (B) Methylation-specific PCR of JunB-M and JunB-U of K562 cells after 5-aza-2′-deoxycytidine treatment. JunB-M PCR products showed a time-dependent decrease of intensities and JunB-U PCR products showed a slight increase of intensities. Internal controls are PCR products of universal primer set JunB-MU. (C) Allelic patterns of CpG site methylation at the promoter area of JunB gene of K562 cells before and 5 days after 5-aza-2′-deoxycytidine treatment. Methylated CpG sites are marked as filled circles (●) and unmethylated sites as open circles (○).