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For scratch wound assays, 24-well dishes were precoated with 10 \u03bcg\/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 \u03b14\u03b21 but not \u03b15\u03b21 integrin bind to recombinant ADAM12 disintegrin domain. A previous study demonstrated that GFP-tagged \u03b14 integrin behaves like untagged \u03b14 integrin (Pinco et al., 2002). Transwell migration assays were performed as described previously (Liu et al., 1999), with the following modifications.<\/p>\n
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 \u03bcg\/ml FN, and number of cells that migrated through the pores were determined after an incubation of 15 h (for CHOB2-\u03b14 and CHOB2-\u03b15 cells) or 4 h (for CHO-\u03b14\/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.<\/p>\n
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(B) CHO-\u03b14\/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 \u03bcg\/ml CS-1. Similarly, expression of ADAM12 in CHO-\u03b14\/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-\u03b14 cells in both transwell (Figure 2A) and scratch wound (Figure 2B) assays.<\/p>\n