GSC induces EMT but not collective cell migration of HCC cells.

(A) Ectopic expression of GSC in Hep3B cells induced changes in expression of EMT markers, particularly E-cadherin. The expression of each protein was normalized to β-actin. Results were analyzed using Student's t test. (*, P<0.05; **, P<0.01) ANOVA analysis also indicated significant differences among the three groups (P = 0.001 for GSC, P<0.001 for E-cadherin, P = 0.004 for β-catenin, P<0.001 for N-cadherin, and P = 0.007 for vimentin). In addition, post hoc analysis confirmed the significant difference between Hep3B-GSC cells and Hep3B cells without treatment or treated with negative control. (B) Downregulation of GSC in Hep3B cells reversed the changes in expression of EMT markers as detected by Western blot. Results were analyzed using Student's t test. (*, P<0.05; **, P<0.01) ANOVA analysis also indicated significant differences among the three groups (P<0.001 for GSC, P<0.001 for E-cadherin, P<0.001 for β-catenin, P<0.001 for N-cadherin, and P<0.001 for vimentin). Furthermore, post-hoc analysis confirmed the significant difference between Hep3B-GSC cells and Hep3B cells without treatment or treated with negative control. (C) The scattering cells (black arrow) in the Hep3B-GSC group were prominently increased compared to the control groups. Continuous counting of seven fields of individual migrating cells showed higher numbers of Hep3B-GSC cells migrating than the Hep3B and the Hep3B-NC groups. (D) Fluorescence micrographs of Hep3B cells expressing GSC or GFP control. Hep3B-GSC cells showed decreased cell-cell contact and strong migration of the individual type (white arrows) under continuous observation. Hep3B-NC cells showed characteristic integrated migration (white contour line). (100× original magnification, size bar: 100 µm).