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Plates were washed twice with PBS to remove unbound cells at designated time points after scratch wounding, and cells were viewed on a phase contrast microscope (Nikon, Melville, NY) and photographed with an attached camera (Diagnostic Instruments, Sterling Heights, MI). The cells were washed twice with phosphate-buffered saline (PBS) and then cultured in media containing 10% FBS in a tissue culture incubator. Briefly, transfected cells were plated onto 24-well tissue culture plates coated with 10 μg/ml human plasma FN (Roche Diagnostics) or GST-CCBD, or 2 μg/ml CS-1. Molt 3 cells, obtained from American Type Culture Collection (Manassas, VA), were maintained in RPMI 1640 medium containing 10% FBS, l-glutamine, antibiotics, sodium pyruvate, and 15 mM HEPES buffer.
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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.
In contrast, lamellipodia protrusions and cell body translocation seemed less robust in ADAM12 transfected samples (Figure 4B, right, and Movie 1B). Confluent cell monolayers were then scratch wounded and observed by videomicroscopy, as described in the legend https://yabancidiziizle.org/ to Movie 1. After 24 h they were analyzed in transwell migration assays as described in the legend to Figure 2, except that the transwell inserts were coated with 2 μg/ml CS-1. Moreover, this pattern correlates with the selective adhesive activity of the ADAM12 disintegrin domain (Figure 1). 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.
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