EphA3 silencing prevents spreading of LK63 cells on ephrin-A5 surfaces. (A) Silencing of EphA3 in LK63 cells by lentivirus-shRNA knockdown was assessed by Western blot analysis of cell lysates. Four different TCR constructs, nos. 9 to 12, were used for the experiment. Cells derived from transfection with TCR no. 9 were further enriched for EphA3^silenced cells by MACS (no. 9*). Relative EphA3 levels in cell lysates were compared by densitometry (bottom panel) using α-actin Western blot as reference. (B) Comparison of EphA3 cell-surface expression in parental and EphA3 knockdown LK63 cells by flow cytometry using Alexa⁴⁸⁸ IIIA4 α-EphA3 mAb. The profile of clone no. 9* transfected cells (light green) is compared with parental LK63 cells (purple) and with HEK293T cells (green) with known, low EphA3 expression.²⁵  The profiles of LK63 cells stained with a nonrelevant, isotype-matched control antibody (dark green), and in the absence of IIIA4 (red), are illustrated as controls. (C) The ability of EphA3^silenced LK63 cells (clone no. 9*), or control lentivirus-transfected cells, to adhere and spread onto ephrin-A5-Fc– or fibronectin-coated surfaces (as indicated) was examined by confocal microscopy. Fixed cells were stained with Alexa⁴⁸⁸-phalloidin and anti-EphA3 antibodies/Alexa⁵⁴⁶ secondary antibodies. White arrowheads indicate the presence of EphA3 clusters on the tips of filopodia-like cell processes in a cell with residual EphA3 expression. Scale bar represents 10 μm. (D) The fraction of adherent LK63 cells on ephrin-A5-Fc–coated (light gray) or fibronectin-coated (dark gray) surfaces characterized by a spread-out phenotype with actin-rich extensions (as illustrated in panel C) was estimated by a blinded observer counting phalloidin-stained cells. Mean and SE, estimated from 10 microscopic fields per group containing 20 to 40 cells each, are shown.