Notably, in cells treated with both GC7 and siEIF5A2 cell migration was almost completely abrogated (Fig. 4D), suggesting that hypusination is essential for EIF5A2 to exert its role in cell motility. Morphological changes were observed in EIF5A2 overexpressing LO2 cells, suggesting

that these cells may undergo EMT. LO2-Vec cells maintained highly organized cell-cell adhesion; however, when plated at the same cell density, LO2-EIF5A2 cells exhibited a cell scattering phenomenon and loss of cell-cell contact, accompanied by the spindle-shaped, fibroblastic morphology (Fig. 5A). To further demonstrate this phenotype in EIF5A2 overexpressing cells, western blot and IF analysis were carried out. In LO2-EIF5A2 cells, expression of E-cadherin and β-catenin decreased, whereas Microtubule Associated inhibitor the expression of α-catenin remained unaltered; on the other hand, all mesenchymal markers tested, including fibronectin, N-cadherin, vimentin, and α-smooth muscle actin were elevated in LO2-EIF5A2 cells. These data reinforced that EIF5A2 overexpression may induce EMT (Fig. 5B,C). The western blotting results were confirmed by IF analysis (Fig. 5D). In particular, vimentin intermediate filaments localized in a concentrated

and polarized pattern in LO2-Vec cells; however, in LO2-EIF5A2 cells a network of vimentin intermediate filaments was clearly visible. In addition, the mesenchymal marker fibronectin was completely undetectable in LO2-Vec cells, whereas LO2-EIF5A2 cells showed positive staining of fibronectin in the cytoplasm. Taken together, Seliciclib these results medchemexpress strongly suggested that EMT may be activated by EIF5A2 overexpression. During tumor

invasion and metastasis, changes in Rho-GTPase activity will lead to subsequent reorganization of actin cytoskeleton, which in turn causes disruption of adherent junctions. Because LO2-EIF5A2 cells displayed morphological alterations, we further investigated whether EIF5A2 could modulate cytoskeleton rearrangement and activation of Rho-GTPase. F-actin staining revealed that stress fiber and lamellipodia were observed in LO2-EIF5A2 cells but not in control LO2-Vec cells (Fig. 6A). Conversely, EIF5A2 knockdown by siRNA resulted in loss of stress fiber in H2M cells (Fig. 6B). These findings suggested that the migratory phenotype induced by EIF5A2 may be associated with activation of Rho-GTPases. To test this possibility, pull-down assays were used to quantify the amount of the GTP-bound active form of RhoA, Rac1, and Cdc42. Despite a higher level of RhoA in control cells, the active form of RhoA could only be detected in LO2-EIF5A2 cells (Fig. 6C). Similarly, a higher level of active Rac1 was observed in LO2-EIF5A2 cells compared with LO2-Vec cells. Cdc42 was barely detectable in either LO2-Vec or LO2-EIF5A2 cells; no active form was observed (Fig. 6C).