FIGURE 6.

RASSF1A promoter methylation sequencing treated A549 cells. A, the hRASSF1A promoter sequence (NM 007182.4) is shown. The top sequence is deaminated DNA with possible methylation sites, highlighted in gray and numbered, aligned with the endogenous sequence on the bottom. ATG start site is highlighted in black with white text. B, A549 cells were treated with 10 μm SW155246 or 6 μm 5-azacytidine for 72 h, with fresh addition of compound at 48 h. The cells were then harvested and genomic DNA was obtained. The genomic DNA was deaminated and the RASSF1A promoter was amplified through PCR. The PCR products were cloned into E. coli and insert DNA was checked using colony PCR. Plasmid DNA was isolated from positive clones and sequenced using Big Dye Terminator chemistry. Sequences were analyzed using Analyst Software. The DNA was confirmed to have complete deamination. Data from 20 clones of each are shown. Conditions and calculations are described under “Experimental Procedures.”