Effects of NFI-C overexpression and inactivation on the KLF4-E-cadherin signaling pathway in MCF7 breast cancer cells. (A, B) Expression of NFI-C, KLF4, Slug, E-cadherin, Vimentin, N-cadherin, and P-cadherin was analyzed by real-time PCR (A) and western blotting (B) in MCF7 cells transfected with NFI-C-expressing or NFI-C-siRNA constructs or treated with TGF-β. (C, D) Transcriptional activity of the KLF4 promoter and the E-cadherin promoter were evaluated by luciferase assays using NFI-C-expressing or NFI-C-siRNA constructs in MCF7 cells. Control samples were transfected with only E-cadherin promoter constructs. Data are presented as the mean ± SD of triplicate experiments. * denotes values significantly different from the control (P < 0.01). (E) NFI-C binding to the KLF4 promoter was investigated using ChIP assays after transfection with NFI-C-expressing or NFI-C-siRNA constructs in MCF7 cells. (F) The NFI-C binding motif in the KLF4 promoter was confirmed by DNAP assay using extracts from MCF7 cells that had been transfected with NFI-C-expressing or NFI-C-siRNA constructs. Biotinylated oligonucleotides corresponding to the KLF4 promoter (WT or mutated) were used as probes. WT, wild type; MT, mutant type.