Collagen I dependent 2D and 3D cell migration is regulated by CD47.

<p>(A) Wound healing assay; Int cells were pre-incubated or not with 20 µg/ml of the CD47 functional blocking antibody B6H12 for 2 hours, IgG or β2 integrin antibody for 20 minutes, or 100 µM of the COX-2 specific inhibitor NS-398 for 30 minutes, thereafter cells were plated onto 10 µg/ml collagen I coated dishes for 2 hours, after which a wound was made in the monolayer, and the cells were allowed to migrate for 18 hours. Pictures of the wound were taken after 0 and 18 hours. The wound closure was measured and is presented as the percentage of wound closure as compared to time zero. (B, C) 3D cell migration assay; (B) Cells were pre-incubated or not with 20 µg/ml of the CD47 functional blocking antibody B6H12 for 2 hours, IgG or the β2 integrin antibody for 20 minutes, or 100 µM of the COX-2 specific inhibitor NS-398 for 30 minutes after which 250,000 cells from each group were allowed to migrate through a 3 mg/ml collagen I gel and across an 8.0 µm micropore membrane for 18 hours. (C) Int 407 cells were transfected with 50 nM siRNA against CD47 (as indicated) or with scrambled control siRNA, for 48 hours. Thereafter 250,000 cells from each group were allowed to migrate through a 3 mg/ml collagen I gel and across an 8.0 µm micropore membrane for 18 hours. Cell migration was examined by staining with crystal violet blue and measuring the absorbance at 590 nm. The data are given as percent of control and represent means ± SE of five separate experiments. The statistical analyses were performed with unpaired Students t-test; *P<0.05, **P<0.01 relative to the control.