Fig. 6

Promoter CpG island-specific methylation alterations coincide with the repression of ZNFX1. A qRT-PCR analysis demonstrating that DAC increases ZNFX1 expression in lung cancer cells not in normal lung epithelia. DAC abrogates CSC-mediated repression of ZNFX1 in normal lung epithelia and lung cancer cells. B MeDIP analysis of DNA methylation profiles in the first CpG island proximal to the TSS of ZNFX1 in SAEC and Calu-6 cells; DAC decreases CSC- or ZFAS1-mediated DNA hypermethylation. C, D Bisulfite sequencing of two genomic regions in the first CpG island of the ZNFX1 promoter, demonstrating that CSC or ZFAS1 overexpression enhances DNA methylation in this CpG island in SAEC and Calu-6 cells. E MSP demonstrating site-specific DNA methylation in the ZNFX1 promoter in lung cancer cells but not in NREC. F MSP analysis demonstrating DNA methylation in the first CpG island of the ZNFX1 promoter in lung cancer tissues from both smokers and nonsmokers relative to normal adjacent lung tissues. Data are mean ± SEM; T-test; n = 3; * p < 0.05; **p < 0.01