Figure 5. Chronic Sunitinib treatment induced AXL and MET dependent expression of VEGF, and promoted HUVEC angiogenesis.

(A) 786-O cells, 786-O cells that stably expressed AXL or MET shRNA constructs were chronically treated with sunitinib (1 μM) and then deprived of sunitinib, and 786-O parental cells were seeded (10, 000 per well) in Matrigel (1:2 diluted) in 96 well plates, HUVEC cells were seeded on top of Matrigel (5, 000 per well). After 24 hours, HUVEC cell tube formation was recorded with a bright field microscope. Data represent three independent experiments. The tube lengths were measured and quantified with Image J software. (B) Chronic sunitinib pre-treated 786-O that were deprived of sunitinib (S786-O) or 786-O parental cells (10, 000 per well) were seeded in Matrigel (1:2 diluted) in 96 well plates, HUVEC cells were seeded on top of Matrigel (5, 000 per well). The cell co-culture was treated with DMSO, sunitinib (+Suni, 1 μM) or cabozantinib (+Cab, 5μM) for 24 hours, HUVEC tube formation was recorded with a bright field microscope. Data represent three independent experiments. The tube lengths were measured and quantified with Image J software. (C) 786-O cells, 786-O cells that were stably expressed with AXL or MET shRNA constructs were chronically treated with sunitinib (1 μM) and then deprived of sunitinib, and 786-O parental cells were cultured as described in Methods. The conditioned media were collected and analyzed for VEGF levels. The results from four independent experiments were assessed for statistical significance (** indicates p<0.01). (D) Chronic sunitinib pre-treated 786-O that were deprived of sunitinib (S786-O) or 786-O parental cells were cultured as described in Methods. The conditioned media were collected and analyzed for VEGF concentration. The resulted from four independent experiments were assessed for statistical significance (** indicates p<0.01). The data in this figure is representative for 3 shRNA constructs designed to target different sequences in MET and AXL.