The 5′ region of Cdk6 is methylated in c-Jun^Δ/Δ cells. (A) Hypermethylation in c-Jun^fl/fl and c-Jun^Δ/Δ cells after 2, 4, 6, and 8 weeks of p185^BCR-ABL transformation (top panel) and in stable c-Jun^fl/fl and c-Jun^Δ/Δ cell lines (bottom panel), was detected by MSP analysis. A visible PCR product indicates the presence of methylated alleles. H.M. indicates BM of a healthy mouse; +Ctrl, control for methylated samples; −Ctrl, control for unmethylated samples. (B) Location of CpG islands within the 5′ region of Cdk6 (gray boxes). CpG sites are shown as horizontal bars, MSP primer-binding sites are shown as arrows, and AP1-binding sites are shown as vertical bars (black). Twenty-four CpG sites were analyzed by bisulfite genomic sequencing in 2 c-Jun^fl/fl and in 2 c-Jun^Δ/Δ cell lines. In total, 20 clones of each genotype were sequenced. Small black boxes indicate methylated CpG sites and white boxes indicate unmethylated CpG sites. (C) A statistically significant difference between Cdk6 methylation in c-Jun^fl/fl and c-Jun^Δ/Δ cells was observed (n = 20; 2-tailed t test, P < .0001). (D) Immunoblot for c-JUN and CDK6 of c-Jun^Δ/Δ and c-Jun^fl/fl cells after 12, 24, 36, and 48 hours of Aza-dC treatment. β-ACTIN served as the loading control. One representative set of data is depicted. (E) Percentage of living cells 12, 36, 60, and 84 hours after Aza-dC treatment of c-Jun^Δ/Δ and c-Jun^fl/fl leukemic cells. Viability was analyzed by propidium iodide staining. (F) Dnmt1, Dnmt3a, and Dnmt3b mRNA levels of c-Jun^fl/fl and c-Jun^Δ/Δ cells were analyzed by RT-PCR (n = 3; 2-tailed t test, Dnmt3b, P = .016). The fold change compared with c-Jun^fl/fl mRNA levels is shown. Results were normalized by comparison with their Gapdh mRNA expression.