Figure 4
(A) Tivozanib inhibits uPA activity. The cells were treated with tivozanib for 48 h and equal amounts of total secreted protein from each sample were subjected to a chromogenic substrate, which is cleaved by active uPA and produces a colorimetrically detectable product. (B) Tivozanib inhibits enzymatic levels of MMP-2. The conditioned media from tivozanib-treated cells was separated on a non-reducing polyacrylamide gel containing gelatin A. Gelatinolytic activities are visualized as clear bands against the blue background of stained gelatin. The concentrations of tivozanib were 2, 5, 10 and 20 μM. The zymograms are representative of three independent experiments with similar results. The gels were cropped and the full-length gels are presented in Supplementary Fig. 3 (C,D) Tivozanib hinders migration and invasion. The cells were placed into 8-μm porous culture inserts, treated with tivozanib and allowed to migrate for 48 h. The migrated cells on the lower surface of the inserts were quantified by crystal violet staining. For invasion assay, the cells were placed into matrigel-coated inserts and allowed to invade through the matrigel layer for 48 h. (E) Tivozanib decreases adhesive potential of the EOC cells. Tivozanib-treated cells were seeded into collagen I-coated culture dishes then the adhesive cells were stained, lysed and the optical densitometry was read. (F) The effect of tivozanib on ICAM-1 expression was measured by qRT-PCR. (G) The cells were treated with tivozanib then protein lysates were subjected to Western blotting and probed with ICAM-1 antibody. β-actin was used as the loading control. The concentrations of tivozanib were 5, 10 and 20 μM. The blots are representative of three independent experiments with similar results. Data are given as mean ± SD. Statistically significant values of *p < 0.05, **p < 0.01, and ***p < 0.001 were determined compared with the control.
