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Figure 5 | BMC Cancer

Figure 5

From: Decreased expression of 17β-hydroxysteroid dehydrogenase type 1 is associated with DNA hypermethylation in colorectal cancer located in the proximal colon

Figure 5

Bisulfite sequencing of the CpG rich region fragment (A) and binding of Pol II to HSD17B1 promoter (B) in HT29 and SW707 colorectal cancer cells treated with 5-dAzaC. HT29 and SW707 cells were incubated for 48 h either in the absence or in the presence of 5-dAzaC (1.00 μM). The cells were then used for genomic DNA isolation followed by bisulfite conversion of cytosine to uracil. The CpG rich region containing 31 CpG dinucleotides (chr17: 37 953 392-37 953 917) was then amplified by a pair of primers complementary to the bisulfite-DNA modified sequence (Additional file 1, Additional file 2). The PCR products were purified with subsequent cloning into a plasmid vector. Plasmid DNA isolated from ten positive bacterial clones was used for commercial sequencing. The results of bisulfite sequencing were assessed and presented using BiQ analyzer software and BDPC web server, respectively [22, 23]. Black and grey boxes represent methylated and unmethylated CpG dinucleotide, respectively. White boxes correspond to undetermined CpG dinucleotide. For the ChIP assay, HT29 and SW707 cells were incubated for 0, 6, 12, 24, 36, and 48 h either in the absence or in the presence of 5-dAzaC (1.00 μM). After incubation, cells were used for ChIP analysis with anti-Pol II Ab. RQ-PCR was carried out by pairs of primers complementary to the HSD17B1 promoter for the HT29 (--) and SW707 (...▲...) cell lines (Additional file 1, Additional file 2). Data are expressed as a percentage of the HSD17B1 promoter occupied by Pol II. The results are presented as the mean ± SE from three independent experiments.

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