FIG. 1.

Bisulfite analysis of DNA methylation in cell lines. Filled black circles represent >80% methylation at a given CpG after sequencing 5 to 10 individual clones. A gray circle represents between 20 and 80% methylation at a given CpG. An open circle represents <20% methylation at a given CpG. (A) Three cell lines were analyzed as representative of cells incapable of producing TNF-α (K562) and competent to produce TNF-α (THP-1 and HL-60). THP-1 cells were stimulated with LPS, sorted by flow cytometry based on expression of TNF-α, and analyzed by bisulfite conversion. These results are indicated as positive (+) and negative (−). (B) Individual clones of two partially methylated cell lines (K562 and THP-1). THP-1 cells show significant heterogeneity in the pattern of methylation between clones, whereas K562 has sporadic bases demethylated on all clones. (C) Flow cytometry demonstrating the cell lines' competence to produce TNF-α. K562 cells (top panel) were stimulated with 100 ng/ml of PMA for 6 h. (K562 does not express TLR4 or CD14 receptors for LPS.) THP-1 cells (lower panel) were stimulated with 1 μg/ml of LPS for 6 h. The black fill represents the unstimulated cells stained with the anti-TNF-α antibody. The gray fill represents the stimulated cells.