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. 2000 Dec 19;98(1):265–270. doi: 10.1073/pnas.98.1.265

Figure 2.

Figure 2

(a) Bisulfite sequencing of HPP1 hypermethylated fragment. HPP1 sequence with CpG sites is indicated (*). (Top) Bisulfite-modified DNA of normal mucosa showing C→T conversion. (Middle and Bottom) Hyperplastic polyp and tumor specimens, respectively, showing methylated (unconverted) CpG sites following bisulfite treatment. (b) COBRA assay. A 427-bp PCR fragment cleaved by RsaI when methylation is present in bisulfite-modified DNA. M, 100-bp DNA ladder (Promega); N, normal mucosa; T, tumor DNA. Histogram showing distribution of COBRA methylation in normal mucosa (N) and tumor (T) samples and illustrating cluster of results below 30% in normal mucosa samples. (c) Differential expression of HPP1 as indicated by the presence or absence of a 307-bp cDNA fragment after RT-PCR in normal mucosa (N) and corresponding tumor (T) samples. (d) Expression of HPP1 in LoVo was restored after 12 h of treatment with the demethylating agent 5-AzaC (0.5 μM) and maintained up to 72 h posttreatment (*). Corresponding β-actin expression for each sample is represented below. M, 100-bp DNA ladder (Promega); B, negative control.