In find more contrast, PIA treatment of the cells seemed to restore their epithelial morphology of a polygonal shape (Fig. 4A upper panel). In phalloidin staining, KOSCC-25B cells demonstrated circumferential, cortical actin, and actin in elongated filopodia; however, no actin stress fibers were detected. In contrast, PIA-treated cells revealed Evofosfamide clinical trial an abudance of actin stress

fibers (Fig. 4A lower panel). These results showed that PIA treatment of the cells induced actin cytoskeleton reorganization, which contributed to loss of the migratory phenotype. We examined whether PIA treatment could affect the expression and localization of E-cadherin and β-catenin, epithelial markers, and Vimentin, a mesenchymal marker. In accordance with the observed morphologic change, inhibition of Akt activity induced the expression in immunoblotting and RT-PCR (Fig. 4B) and localization of E-cadherin

and β-catenin as seen in the immunofluorescence analysis (Fig. 5 upper and middle panel). Also, PIA treatment decreased the vimentin expression (Fig. 4B) or localization (Fig. 5 lower panel), although the change was not as prominent as that in the epithelial markers. Figure 4 Effects of Akt inhibition on cell morphology and the expression of the epithelial and mesenchymal markers. (A) KOSCC-25B cells had an Blasticidin S mw elongated shape, assuming a fibroblast-like appearance. In contrast, PIA-treated KOSCC-25B cells seemed to restore their epithelial morphology of a polygonal shape. In phalloidin staining, KOSCC-25B cells demonstrated circumferential, cortical actin (blue arrowheads), and actin in elongated filopodia (white arrowheads); however, no actin stress fibers were detected. In contrast, PIA-treated cells revealed an abudance of actin stress fibers (yellow arrowheads). Scale bar: tetracosactide 100 μm (black), 20 μm (white). (B) Inhibition of Akt activity increased the expression of E-cadherin and β-catenin, and reduced the Vimentin expression in KB and KOSCC-25B cells.

Figure 5 Effects of Akt inhibition on the localization of the epithelial and mesenchymal markers. The inhibition of Akt activity induced the localization of E-cadherin and β-catenin, and decreased that of vimentin, as seen in the immunofluorescence analysis. Reduced migratory ability after Akt inhibition In order to examine whether inhibition of Akt activity could affect cell motility, we performed an in vitro migration assay. The numbers of KB and KOSCC-25B cells from the PIA-treated group that migrated through the filter were only 61.1% and 56.4% of that in control cells (P < 0.05; Fig. 6), respectively. Figure 6 Reduced migratory ability due to Akt inhibition. Photomicrography of control (A) and PIA-treated (B) KOSCC-25B groups in the in vitro migration assay. (C) The numbers of KB and KOSCC-25B cells from the PIA-treated group that migrated through the filter were only 61.1% and 56.