(A) Western blot analyses showed acquisition of mesenchymal markers and loss of epithelial marker proteins in the CTHRC1 transfectants compared with vector control cells. FN, fibronectin; VIM, vimentin; α-SMA, alpha smooth muscle actin; N-cad, N-cadherin; E-cad, E-cadherin; CK, cytokeratin; Des, desmoplakin. (B) Snail expression and CREB activation were demonstrated in HepG2 cells stably expressing CTHRC1, other transcription factors Slug, Twist1 and Twist2 were not changed. (C) Snail expression and CREB activation were also observed in HLK-3 and HepG2 cells transduced with CTHRC1 expressing adenovirus for 48 hours. (D) MMP9, but not MMP2, was up-regulated by transient transfection with Snail in HepG2 cells. (E) Conditioned medium was collected and concentrated by centrifugation. Zymogram gel assays was performed. The gelatinolytic activity of MMP2 and MMP9 was assessed by Coomassie blue staining. The protein levels of MMP2 and MMP9 in stable CTHRC1 transfectants, compared with vector control cells checked by Western blotting. (F) MMP2 and MMP9 activity and expression from the HLK-3 and HepG2 cells transduced with AdCTHRC1 or AdLacZ were measured by zymogram gel assays and Western blotting.