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. 2008 Nov 27;100(1):117–125. doi: 10.1111/j.1349-7006.2008.01017.x

Figure 5.

Figure 5

The expressions of tumor suppressor genes are decreased through DNA methylation by constitutively active RAF (caRAF). (a) 5‐aza‐dC restores the mRNA transcription of p16INK4A , p21WAF1 and p27KIP1 in caRAF‐GES‐1. (b) The promoters of p16INK4A , p21WAF1 and p27KIP1 were methylated in caRAF‐GES‐1 cells. Primers of specific for unmethylated (U) or methylation (M) DNA were used to amplify DNA from caRAF‐GES‐1, or treated with 25 µM PD98059, or with 5‐aza‐dC (2 µM). (c) Bisulfite sequencing chromatogram of p16INK4A and p21WAF1 CpGs of p16INK4A in caRAF‐GES‐1 cells show numerous thymidines due to sodium bisulfite conversion of unmethylated cytosines to uracil. Whereas GES‐1 cells (with empty plasmid transfection) or the caRAF‐GES‐1 cells (treatment with 5‐aza‐dC or PD98059) show multiple cytosines due to the resistance of methylated cytosines within CpG dinucleotides to bisulfite conversion. The sequencing of p21WAF1 shows that not all methylated cytosines within CpG dinucleotides are abolished by PD98059. Both sequences are compared to the wild‐type (WT) genomic DNA sequence. DMSO, dimethylsulfoxide.