Figure 4. Cabozantinib suppressed MET/AXL signaling and chronic sunitinib treatment induced EMT.

(A) Chronic sunitinib treated 786-O cells and 786-O parental cells were cultured in medium with or without sunitinib (1μM) for 3 days, and then treated with or without cabozantinib (5μM, 24h) as indicated. The cells were lysed and examined by western blot with specific antibodies as indicated. Data represent three independent experiments. (B) Chronic sunitinib treated 786-O cells were cultured in medium without sunitinib for 3 days and then treated with cabozantinib (5μM, 24h) as indicated in serum free medium. The cells were then stimulated by HGF (100ng/ml, 30minutes) or GAS6 (200ng/ml, 30 minutes). The cells were then lysed and examined by western blot with specific antibodies as indicated. Data represent three independent experiments. (C). Chronic sunitinib treated 786-O cells and 786-O parental cells were cultured in medium without sunitinib before experiment. The cells were then seeded for wound-healing experiment as described in Methods. The cells were treated with DMSO or cabozantinib (5μM, 24h) as indicated. The gap distances in four independent experiments were measured and analyzed. (D). The transwells were coated with Matrigel as described in Methods. Chronic sunitinib pre-treated 786-O cells that were deprived of sunitinib (S786-O) or 786-O parental cells (10, 000 per well) were seeded in transwells in serum free medium supplied with cabozantinib (Cab, 5μM) or DMSO as indicated, Complete Growth Medium with cabozantinib (5μM) or DMSO was added to the bottom wells. After 24 hours, the invaded cells were stained and counted as described in Methods. Data represent four independent experiments.