Fig. 8. The phenotype of MK3^– cells is reversed by reconstitution with human 3A. (A) MK3^– cells were transduced with the cDNA encoding human 3A, and cell surface expression was verified by FACS analysis using monoclonal antibody J143 against human 3. Negative control (secondary antibody only) is indicated by thin black line. (B) MK3^R cells were seeded on Ln-332 (left panel) or Col-I (right panel), allowed to spread, and then incubated for 3 hours with GoH3 (10 µg/ml). Scale bar: 20 µm. (C) The number of spread cells was scored and expressed as a percentage of the total number of cells. In each independent experiment, approximately 500 cells per condition were counted. The graphs depict the averages of three experiments (^***P<0.0005). (D) Lysates of MK3⁺, MK3^– and MK3^R cells were analyzed by SDS-PAGE, and expression of integrin 3 was determined by western blotting using mAb 29A3 recognizing both human and murine 3 (L, light chain). Confluent MK3⁺, MK3^–, and MK3^R cells were deprived of growth factors, incubated for 2 hours with mitomycin C (10 µg/ml), and scratched with the tip of a pipette prior to EGF stimulation. Wound areas were determined using Image J, and the ratio of the wound area after overnight migration over the wound area at t=0 was calculated and expressed in a bar graph. Values shown represent the means ± s.e.m. of three independent experiments (^*P<0.05, ^***P<0.0005).