OLFM4 promoter CpG methylation status in AML patients and effect of 5-aza-2′-deoxycytidine on OLFM4 transcription in HL-60 cells. (A) Methylation status (percentage) of 8 CpG sites (−681, −666, −562, −486, −446, −91, +4, and +34, relative to the OLFM4 transcription start site) in the OLFM4 promoter in the DNA isolated from the peripheral blood leukocytes of AML patients. Methylation levels in AML patients with low OLFM4 mRNA expression (L, n = 7), AML patients with high OLFM4 mRNA expression (H, n = 5), and normal individuals (N, n = 10) were measured with pyrosequence. (B) Methylation (percentage) of the 8 CpG sites in the OLFM4 promoter of HL-60 cells was determined with pyrosequence. Each bar (from left to right) in every CpG site group represents 5-aza-2′-deoxycytidine (5μM) treatment for days 0, 1, 2, 3, 4, and 5. Medium was replaced with new medium with freshly added 5-aza-2′-deoxycytidine every 48 hours. (C) HL-60 cells were treated with 5-aza-2′-deoxycytidine (Aza, 5μM) or vehicle for 5 days. Medium was replaced with new medium with freshly added 5-aza-2′-deoxycytidine or vehicle every 48 hours. OLFM4 expression was determined by qRT-PCR. Values relative to β-actin represent the mean ± SD of 3 experiments performed in triplicate. (D) Promoter activity of the 5′-flanking region of the OLFM4 gene (−969 Luc) in HL-60 cells transfected with OLFM4 promoter-reporter constructs that were treated with or without Sss I CpG methylase. 0 Luc represents parental luciferase reporter construct without the OLFM4 promoter. Data represent the relative activities to the Renilla luciferase activities of phRL-TK, which was transfected together with each OLFM4 plasmid construct. Values represent the mean ± SD of 3 experiments performed in triplicate. *P < .05, compared with unmethylated promoter.