FIG. 1.

Responses of HeLa and HCT116 cells to the DNA methylation inhibitor 5-azadC and the role of DNMT1 and DNMT3B. (A) Effect of increasing concentrations of 5-azadC on DNMT1, DNMT3A, and DNMT3B protein levels. HeLa cells were mock treated (−) or treated with 0.1, 0.5, 1.0, 5.0, and 10.0 μM 5-azadC for 48 h (indicated by the wedge); then soluble protein extracts were prepared and subjected to SDS-PAGE followed by Western blotting with the antibodies indicated at the left. GAPDH served as a loading control. (B) Effect of 5-azadC on cell viability. Cells were treated with indicted doses of 5-azadC for 48 h (fresh drug added every 24 h); the medium was changed, and then cell viability was determined using the MTT assay. (C) 5-azadC treatment results in decreased clonogenic survival. Cell lines were treated with 5-azadC for 48 h, followed by replacement of the medium and continued growth for 10 to 12 days. Results are presented as the average of quadruplicate measurements, and the bar is the standard deviation. (D) Effect of increasing doses of 5-azadC on the cell cycle summarized as the relative increase in the number of cells (n-fold) arrested in G₂/M for each of the cell lines following drug treatment. Experiments were repeated three times and averaged. (E) HeLa cells treated with 10 μM 5-azadC for 48 h preferentially arrest in G₂ as determined by DNA content (PI staining shown on the x axis) and staining cells with an antibody for the M phase specific marker phospho-Ser10 histone H3 (y axis) followed by flow cytometry.