EphA3-mediated LK63 cell adhesion is independent of VCAM and ICAM functions. (A) LK63 cells were cocultured with parental HEK293T cells or ephrin-A5–expressing HEK293T clones (ephr-A5/HEK293T) as indicated; the inset is a magnification of the boxed section in the middle panel. Soluble monomeric ephrin-A5 was added to one of the cultures at 100-fold molar excess as competitive inhibitor of cell-surface ephrin-A5. (B) LK63 cells were cocultured on a layer of ephrin-A5/HEK293T cells in Matrigel. After 12, 24, and 48 hours, cells were imaged by bright-field microscopy. Individual LK63 cells (white arrowheads) remain bound to the ephrin-expressing cells to form large coherent colonies (black arrowhead) after 48 hours of culture. (C) Adhesion of LK63 cells to ephrin-A5/HEK293T cells was monitored in the presence of α-VCAM and α-ICAM antibodies. VCAM and ICAM expression on adherent LK63 cells was assessed by staining with α-VCAM and α-ICAM (inset) antibodies and Alexa⁴⁸⁸-conjugated secondary antibodies (left). Following 60-minute coculture in the presence of α-VCAM antibodies alone (middle), or together with α-ICAM antibodies (right), the cytoskeleton of fixed cells was stained with rhodamine-phalloidin. α-VCAM and α-ICAM were detected with Alexa⁴⁸⁸ secondary antibodies; the merged images are shown. (D) LK63 cells were cocultured on a monolayer of untreated (left) or LPS-treated (middle) HMVECS. To affect LPS-induced cell adhesion, HMVECS were incubated with function-blocking α-VCAM and α-ICAM antibodies (right) as indicated. Merged microscopic images (Alexa⁴⁸⁸, green; rhodamine-phalloidin, red) are shown. Scale bars represent 20 μm. (E) Cell-cell adhesion was quantified by counting LK63 cells remaining attached to untreated, α-ICAM-1/α-VCAM–treated or control IgG-treated ephrin-A5/293 cells (□), or untreated, LPS or LPS and α-ICAM-1/α-VCAM treated HMVECs (■) in a minimum of 4 representative microscopic sections at 10× magnification. Mean cell number and SE are shown.