FIGURE 2.

A–C, ADAM-10, but not ADAM-17, knockdown inhibited DN30-induced shedding of pre-Met. Representative Western blots (of three independent experiments) for pre-Met and Met, ADAM-10, and ADAM-17 in A549 (A), SKOV3ip (B), and GTL-16 (C) cells, cultured 5 h with or without 80 μg/ml DN30 (A–C) are shown. α-Tubulin was used to normalize protein levels. Knockdown cell lines (shADAM-10 or shADAM-17, respectively) and control cell lines (shNT) were generated using shRNAi. Densitometric analysis: A: A549shNT without DN30, 100%; with DN30, 48.0% ± 8.2%; n = 3; shADAM17 without DN30, 100%; with DN30, 56.3% ± 8.1%; n = 3; shADAM10 without DN30, 100%; with DN30, 103.7% ± 2.6%; n = 3. B, SKOV3ipshNT without DN30, 100%; with DN30, 38.1% ± 12.7%; n = 3; shADAM17 without DN30, 100%; with DN30, 51.6% ± 14.3%; n = 3; shADAM10 without DN30, 100%; with DN30, 105.3% ± 4.6%; n = 3. C, GTL16shNT without DN30, 100%; with DN30, 63.4% ± 4.2%; n = 3; shADAM17 without DN30, 100%; with DN30, 55.2% ± 5.6%, n = 3; shADAM10 without DN30, 100%; with DN30, 90.8% ± 9.2%, n = 3. Furthermore, in SKOV3ip (B) and GTL-16 cells (C), elevated levels of ADAM-17 after DN30 administration and increased ADAM-10 protein upon knockdown of ADAM-17 were detected. D, representative Western blot of supernatants and immunoprecipitated protein from supernatants of GTL-16shNT or GTL-16shADAM-10 cells are shown, cultured for 5 h with 80 μg/ml DN30. Knockdown of ADAM-10 reduced Met shedding. E, representative Western blot of A549 cells of three independent experiments cultured with or without 80 μg/ml DN30 and the indicated amounts of GI254023X, showed that ADAM-10-specific inhibition by GI254023X abolished Met shedding. Densitometric analysis: without DN30 without GI254023X, 100%; with DN30 without GI254023X, 42.7% ± 5.6%; with DN30 1 μm GI254023X, 46.6% ± 12.8%; with DN30 3 μm GI254023X, 81.8% ± 33.2%; with DN30 5 μm GI254023X, 125.1% ± 28.2%. n.s., not significant.