Wnt3a overexpression induced mesenchymal phenotype and increased expression of mesenchymal markers. (A) Wnt3a protein levels were significantly increased in clone7 and clone15 HCT116 cell pools transfected with Wnt3a plasmid. Then, c-myc and CyclinD1 protein expression increased in clone7 and clone15 HCT116 cell. (B) Immunofluorescent staining of F-actin. Wnt3a-overexpressing cells exhibited dramatic changes in cell morphology from a tight packed, polarized, and epithelial-like appearance to a scattered, irregular, and fibroblastic-like shape. (C) Wnt3a-overexpressing cells showed lower E-cadherin expression but higher vimentin expression. β-Catenin expression did not markedly change in three groups of cells. EMT regulatory proteins including Snail, Slug, and Twist were detected. Snail was upregulated in Wnt3a-overexpressing cells compared with control cells, whereas Slug and Twist expression did not significantly change. (D) Immunofluorescent staining of E-cadherin, vimentin, and β-catenin. Wnt3a-overexpressing cells showed lower E-cadherin expression and higher vimentin expression. More β-catenin accumulation in nucleus was observed in Wnt3a-overexpressing cells than in control cells. A green or red signal represents staining for corresponding protein, whereas a blue signal represents nuclear DNA staining by 4′,6-diamidino-2-phenylindole.