PTK7 expression and its role in vascular endothelial cell migration. (A) Expressions of Flt-1 and KDR mRNA were determined in short hairpin RNA for Flt-1(shFlt-1) or KDR (shKDR) transduction conditions by real-time RT-PCR. (B) shRNA-transduced cells were plated at a density of 2 × 10⁴/well in a 6-well plate and cultured with VEGF-A (20 ng/mL). Then cell numbers were counted on each time point (**P < .01 vs shNeg). (C) Cell migration after wound breakage was determined by cell restitution assay. Some shFlt-1–transduced cells were transfected with PTK7-silencing siRNA (○). VEGF-A (20 ng/mL) was added to serum-free medium and cell migration was observed over the next 36 hours. Migratory cells were counted using inverted phase-contrast microscopy (×100; *P < .05, **P < .01). (D) PTK7 expression changes in healing wound breaks were measured by immunoblotting. After wound breaks were created by pipette tips in confluent cell layers, cell samples were serially collected over the next 36 hours and immunoblotting was performed as described. Band densities were measured and statistically analyzed (*P < .05, **P < .01). (E) Cells were stained using anti–mouse PTK7 monoclonal antibody during the wound break assay. MS1 cells were stained 6 hours (left and middle panels) and 18 hours (right panel) after wounding. Enhanced localized PTK7 expression was observed in the wound margin (white arrow; ×200, except bottom right [×400]; green: PTK7; blue: DAPI). Arrowhead indicates lamellipodia and filopodia staining with PTK7 antibody.