Down-regulated γ-catenin expression is associated with tumor aggressiveness in esophageal cancer

Figure 2 Alterations of cell-cell adhesion and cell migration exerted by RNAi-mediated knockdown of γ-catenin. A: Representative Western blot of γ-catenin in 4 esophageal cancer cell lines; B: KYSE150 and TE3 cells were transfected with γ-catenin siRNAs (siRNA-1 and -2), or a negative control siRNA (control). Western blot analysis was used to show RNAi-mediated knockdown. Equal loading was ascertained using β-actin as an internal control; C: Hanging drop assay. Cells were seeded into hanging drop cultures and allowed to aggregate for 24 h. After trituration by passing the cell cluster 30 times through a 200 μL pipette tip, the degree of dissociation of the cell cluster was visualized by microscopy and quantified by manual counting under a dissecting microscope; D: Dispase-based dissociation assay. Cell monolayers were separated from culture dishes via incubation with dispase. Monolayers were transferred to 15 mL conical tubes containing 2 mL of phosphate buffered saline. After 50 inversions, the degree of fragmentation of the monolayers was observed. The dissociation assay was quantified by counting the number of total particles; E: Transwell assay was performed to determine cell migration. Migrated cells were fixed and stained, and representative fields were photographed. The cells were quantified in 10 random fields with a light microscope (× 200). The mean value was calculated from data obtained from three separate chambers. ^bP < 0.01 vs the control group.