Figure 8. (A and B) High-resolution melting (HRM) analysis of the indicated cell lines. Genomic DNA of each sample was sodium bisulfite-treated, amplified, and melted as described in “Materials and Methods”. (C and D) Pyrosequencing analysis: on the top of each panel the pyrosequenced regions in the miR200 promoter on chromosome 1 (C) and on chromosome 12 (D) are indicated. Numbers indicate the distance from the transcription start site. Vertical bars are the position of the Cs that can be methylated in each sequence. The mean methylation % of each pyrosequenced region of the indicated cell lines untreated or treated with TGF-β (5 ng/ml) is specified in the histograms (C and D). Each experimental point was pyrosequenced 3 times. Data are reported ± standard deviation. (E and F) Relative miRNA levels were measured by qRT-PCR in the indicated cell lines untreated or treated with TGF-β. miR200b, miR200a, and miR429 encoded by the cluster on chromosome 1 are shown in (E), miR200c and miR141 on chromosome 12 are in (F). (G) Analyses of mRNA levels of c-myb and ZEB1 were performed by qRT-PCR in the indicated cells untreated or treated with TGF-β. d, days. qRT-PCR data are reported ± standard deviation.