Fig.2. Overexpression of NUMB-1 in KYSE150 cells inhibits cell motility and EMT induced by EGF.

(A) A wound was introduced in sub-confluent cultures of KYSE150 cells that either overexpressed NUMB-1 (Ad-NUMB-1) or a control vector (Ad-Null). Wound closure was monitored at the indicated time points, and a representative image from three independent experiments is shown (original magnification, 200×). (B) Invasiveness of the cells was analyzed in a Boyden chamber that was either coated with Matrigel or left uncoated. Cells that invaded through the filter and adhered to the lower surface of the filter were stained and analyzed by microscopy. Results are shown as mean ± SD (* p<0.01). (C) The cellular morphology of control KYSE150 cells and NUMB-1-overexpressors, cultured on plastic and treated with various concentrations of EGF for 48 h after a 24h starvation period, was observed by phase contrast microscopy. The control KYSE150 cells (top panel) exhibit a spindle-like, fibroblastic morphology, while NUMB-1-overexpressing cells (bottom panel) retain a cobblestone-like appearance even upon treatment with high doses of EGF. Representative images were acquired at the time points indicated in the top left corner of each image (original magnification, 400×). (D) The expression of E-cadherin, Vimentin, and α-SMA upon EGF treatment in control cells and NUMB-1 overexpressing KYSE150 cells was analyzed by Western blotting.