Axitinib Has Antiangiogenic and Antitumorigenic Activity in Myxoid Liposarcoma

Figure 4

Characterization of axitinib. MLS 402 and MLS 1765 cells were treated with IC₅₀ axitinib (MLS 402, 1.2 μM; MLS 1765, 3.2 μM), imatinib (10 μM), or vehicle control for 2 h, lyzed, and analyzed for anolytes targeting the phosphorylation site of PDGFRα, PDGFRβ, and c-Kit. The experiment was performed 3 times, and the data are presented as mean + SEM. t-AKT, total AKT (a). MLS 1765 cells were treated overnight with IC₅₀ axitinib or vehicle control and immunoprecipitated for total VEGFR3 (t-VEGFR3) and phospho-VEGFR3 (p-VEGFR3). Densitometry of total VEGFR3 expression and phospho-VEGFR3 expression following axitinib treatment (b). MLS 402 and MLS 1765 cells were treated with IC₅₀ axitinib or vehicle control for 2 h, and then the cells were lyzed and analyzed for phospho-AKT and phospho-ERK1/2 by Bio-Plex. Three technical and biological replicates were tested. The data are presented as the mean + SEM (c). MLS 402 and MLS 1765 cells were treated overnight with IC₅₀ axitinib or vehicle control, and then cells were analyzed by RT-qPCR for the expression of VEGFR1, VEGFR3, VEGFA, and VEGFB. The experiment was performed three times, and the data are presented as the mean + SEM (d). HUVECs were suspended in Matrigel and then were treated with conditioned medium from cells that had been pretreated with IC₅₀ axitinib or vehicle control. Images were acquired at hourly intervals, and the figure displays representative images taken at 8 h. Tube lengths were measured using ImageJ (version 1.47d). The experiment was performed three times, and the data are presented as the mean + SEM (right) (e). Ax, axitinib; CM, conditioned medium; IB, immunoblotting; Im, imatinib; IP, immunoprecipitation; VC, vehicle control; LO, Lipofectamine-only treated cells; Ut, untreated MLS cells. A paired, two-tailed -test was performed. ; ; ; .

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