Figure 5
NKX6.1 represses cancer invasion by increasing the expression of epithelial markers. (a) Representative images of the morphology of HeLa cells expressing NKX6.1 or the vector only at low (upper image) and high (lower image) cell density (original magnification, × 100). (b) The expression of EMT regulators was detected by western blotting in NKX6.1-expressing HeLa cells. (c) The expression of E-cadherin was analyzed in NKX6.1-expressing cells and NKX6.1-shRNA-knockdown cells by qRT–PCR (bar graphs) and western blotting (below). (d) IF assays were used to detect E-cadherin expression in NKX6.1-expressing CaSki cells. (e) The upper scheme depicts WT (pGL4.21-E-cadherin) and HDBS-mutated (Mut; pGL4.21-E-cadherin) E-cadherin promoters. The activity of different E-cadherin promoter constructs in HeLa cells was analyzed by a luciferase reporter assay. (f) Chromatin from HeLa cells expressing NKX6.1 or SiHa cells expressing NKX6.1 shRNAs was immunoprecipitated with indicated antibodies and then analyzed by quantitative PCR using E-cadherin-specific primers. The upper scheme depicts the position of the HDBS within the promoter region of E-cadherin. (g) HeLa cells were transfected with the indicated combinations of vectors, and Matrigel invasion assays were used to analyze the effects on cancer invasion. Two specific E-cadherin shRNAs (nos 1 and 2) were used. The numbers in the western blots represent the ratios of targets to the internal control. The data are presented as the mean±s.e. from three independent experiments in triplicates (analyzed by unpaired two-tailed t-test). *P<0.05, **P<0.01 and ***P<0.001.
