CBX8 directly binds to and down-regulates the Snail promoter activity. (A, B) The indicated stable cell lines were transfected with Snail or Slug promoter linked to luciferase for 48 h and then subjected to the luciferase assay as described in Materials and Methods. (C) The upper panel, a schematic illustration of the Snail promoter regions (1-16) with (+) or without (-) binding affinity for CBX8. The arrow indicates the transcriptional start site (TSS). The lower panel, the result of ChIP-quantitative PCR analysis of CBX8 binding to the distinct regions in the Snail promoter, showing enrichment with CBX8 antibody compared with IgG control. The p16 and GAPDH promoters were used as the positive and negative controls, respectively. The results are expressed as the mean ± SD of individual samples from three independent experiments. ** p < 0.01 and *** p < 0.001, Student's t-test. (D) There was a reverse correlation between CBX8 and Snail at mRNA level in ESCC tissue samples. p = 0.0215, linear regression analysis.