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For scratch wound assays, 24-well dishes were precoated with 10 μg/ml FN, and migration assays were conducted as described in Materials and Methods. Cells were either mock transfected or transfected to express full-length ADAM12, and transwell migration assays (A, C, E, and G) or scratch wound assays (B, D, F, and H) were conducted 24 h after transfection. Cells expressing α4β1 but not α5β1 integrin bind to recombinant ADAM12 disintegrin domain. A previous study demonstrated that GFP-tagged α4 integrin behaves like untagged α4 integrin (Pinco et al., 2002). Transwell migration assays were performed as described previously (Liu et al., 1999), with the following modifications.
For each experiment, the average migration rate of ADAM12-transfected cells was normalized to that of mock-transfected cells. For transwell migration assays, the distal membranes of transwell inserts were coated with 10 μg/ml FN, and number of cells that migrated through the pores were determined after an incubation of 15 h (for CHOB2-α4 and CHOB2-α5 cells) or 4 h (for CHO-α4/GFP and CHOK1 cells). We next tested the effect of expressing full-length ADAM12 on directed cell migration on FN, using both transwell and scratch wound assays.
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(B) CHO-α4/GFP cells were transfected as indicated and then analyzed in scratch wound assays, as described in the legend to Figure 2, except that 24-well dishes were coated with 2 μg/ml CS-1. Similarly, expression of ADAM12 in CHO-α4/GFP cells caused a reduction in cell motility in both transwell (Figure 2C) and scratch wound (Figure 2D) assays. Expression of ADAM12 reduced the motility of CHOB2-α4 cells in both transwell (Figure 2A) and scratch wound (Figure 2B) assays.
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