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. 2020 Jan 8;12(1):148. doi: 10.3390/cancers12010148

Figure 3.

Figure 3

Snail1 enhances DNA methylation of the USP4 promoter and suppresses USP4 expression. (A) Promoter region of USP4 was analyzed for Snail1 binding sites. The putative binding sites are underlined. (B) Promoter region of USP4 was analyzed for GC rich (or CpG-rich) regions (potential methylation sites). The histogram was adopted from the MethPrimer website. (C) Illustration of Snail1 binding sites and methylation regions in the USP4 promoter region. (D) The binding sites of the USP4 promoter required for transcriptional suppression by Sanil1 were identified by luciferase assay. HEK293 cells were co-transfected with expression vectors for Snail1 and a luciferase reporter gene controlled by wild type (wt) mutated USP4 promoter sites. (E) DNA methylation of the USP4 promoter region in the presence and absence of Snail1 as determined by bisulfite sequencing PCR. (F,G) Expression levels of USP4 mRNA (F) and protein (G) in snail1-overexpressing D121, LLC, and H1299 cells. Data presented as mean ± SD of three independent experiments (E,F). ** P < 0.01.