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. 2012 Nov 26;7(11):e48954. doi: 10.1371/journal.pone.0048954

Figure 4. DNMT activity is increased in E6 expressing cells and E-cadherin can be restored following treatment with the DNMT inhibitor, 5-Aza-dC.

Figure 4

(a) Nuclear extracts from HCT116 and HCT116 E6 cells were assayed for DNMT activity. n = 3 independent experiments. Error bars represent ± SEM. * P<0.05. (b) RNA was harvested from HCT116 and HCT116 E6 cells treated with 2.5 µM and 7.5 µM 5-Aza-dC for 72 h and analysed by qRT-PCR for E-cadherin mRNA levels, normalized to GAPDH. n = 3 independent experiments. Error bars represent ± SEM. * P<0.05; ns = not significant. (c) Lysates from HCT116 and HCT116 E6 cells treated with 5-Aza-dC, as above, were tested for E-cadherin expression by western blot. Data representative of three experiments. (d) Bisulfite-converted DNA from HCT116 and HCT116 E6 cells was prepared and sequenced to determine the methylation state of CpGs within the minimal promoter region (−191 to +94 bp relative to transcription start site). Nucleotide sequences of ten individual clones of bisulfite converted DNA from each cell line were analysed.