CA9 is upregulated by ZEB1 in tongue cancer cells. (A) A schematic representation of ZEB1 binding sites with the E-box sequence (CACCTG) in the 3kb putative CA9 promoter. The first base of the 3kb strand is defined as ‘1’. (B) Chromatin immunoprecipitation assays identified ZEB1 binding sites within the putative CA9 promoter. Primers specific for sites B, C and E yielded PCR reaction products from ZEB1–DNA immunoprecipitates. The input represents DNA directly after lysis. The PCR reaction product for immunoprecipitates obtained using the RNA Polymerase antibody represents the positive control. (C and D) Changes in CA9 mRNA and protein expression following the inhibition of ZEB1 in tongue cancer cell lines were evaluated by qRT-PCR and western blotting, respectively. (E) Luciferase activity driven by the putative CA9 promoter was higher in Tca8113/PYM cells (which exhibit endogenous ZEB1 overexpression) than in Tca8113 and SCC-25 cells. (F) Reporter assays revealed changes in luciferase activity after inhibition of ZEB1 expression in tongue cancer cells. (G) ZEB1 promoted luciferase activity driven by the putative CA9 promoter in HEK283T cells. * p < 0.01.