Figure 3. α-N-catenin inhibits migration, invasion, and angiogenesis in vitro.

(A) SK-N-AS cells were plated in collagen type I-coated upper chamber of transwell (1x10⁵ cells/well) in serum-free media with the bottom well containing 10% FBS RIPM medium. After 6 h incubation, cells were fixed with 4% paraformaldehyde, stained with DAPI, and migrated cells were counted. Alpha-N-catenin overexpression resulted in a significant reduction in cell migration to 43% of CON cells (^* = p < 0.05 vs CON). (B) Cells (1.5×10⁵/well) in serum-free media were added to the upper well coated with Matrigel on the transwell filters and 10% FBS containing RPMI media added into the bottom well. After 48 h incubation, cells were fixed with 4% paraformaldehyde, stained with DAPI, and invaded cells were counted. Alpha-N-catenin overexpression resulted in a significant reduction in cell migration to 24% of CON cells (^* = p < 0.05 vs CON). (C) HUVECs were cultured in conditioned media collected from SK-N-AS cells transfected with a control vector and CTNNA2 overexpression vectors on Matrigel-coated 24-well plates for 6 h. Tubule staining was performed in triplicate and quantified. The number of tubules formed with HUVECs in conditioned media from α-N-catenin overexpressed cells was decreased to 19.5% of control. (Mean ± SEM; ^* = p < 0.005 vs. CON). (D) IL-8 secretion was measured in conditioned media from cells. Cells (3 × 10⁵ cells/ well) were plated in 6-well plate and cultured for 24 h, then the supernatants were collected for measuring IL-8 secretion by ELISA. α-N-catenin overexpression significantly inhibited IL-8 secretion down to 29.7% of control cells in conditioned media. Data represent mean ± SEM; ^* = p < 0.05 vs. CON. (E) HUVECs were cultured in conditioned media collected from BE(2)-C cells transfected with a control vector and CTNNA2 overexpression vectors on Matrigel-coated 24-well plates for 6 h. Tubule staining was performed in triplicate and quantified. The number of tubules formed with HUVECs in conditioned media from α-N-catenin overexpressed cells was decreased to 34.2% of control. (Mean ± SEM; ^* = p < 0.005 vs. CON).