FIGURE 5.

Effects of SW155246 on in vivo methylation. A, HeLa cells were treated with varying concentrations of compound SW155246 for 72 h. The cells were then harvested and genomic DNA was obtained. The genomic DNA was hydrolyzed and samples were run on a Sciex 3200 Qtrap LC-MS/MS to quantify the amount of unmethylated cytosine and methylated cytosine as compared with labeled standards of each. Each point represents averages from two independent experiments. Asterisks denote a statistically significant difference compared with the untreated HeLa cells. Statistical significance was calculated by the unpaired t test (*, p < 0.05; **, p < 0.01). Conditions and calculations are described under “Experimental Procedures.” B, RASSF1A mRNA expression in cells by real-time RT PCR. A549 and HeLa cells were treated with 6 μm 5-AZA or 10 μm SW155246 for 72 h, with fresh addition of compound at 48 h. The cells were then harvested, RNA was extracted, and cDNA was synthesized. Real-time RT-PCR was performed. Each sample was normalized to RNASE P expression. The experiment was performed in biological triplicate with real-time RT-PCR of samples also done in triplicate. Error bars represent the mean ± S.E. for each sample. Conditions and calculations are described under “Experimental Procedures.”