Antivascular activity of plinabulin. (A) HUVECs were treated with plinabulin (5nM) for 12 hours and assessed for in vitro vascularization with the use of matrigel capillary-like tube structure formation assays (magnification, 4×/0.10 NA oil; media, EBM-2). (Left) Micrograph images show the effect of plinabulin on capillary tube branch formation. (Right) The bar graph represents quantification of capillary-like tube structure formation in response to plinabulin. Branch points in several random view fields/well were counted; values were averaged; and statistically significant differences were measured with the Student t test. (B-C) For migration assay, HUVECs and MM cells were treated with plinabulin (5nM and 10nM) for 12 hours; cells were > 90% viable at this time point. Cells were washed and cultured in serum-free medium, plated on a fibronectin-coated polycarbonate membrane in the upper chamber of trans-well inserts, and exposed for 2 hours to serum containing medium in the lower chamber. Cells migrating to the bottom face of the membrane were fixed with 90% ethanol and stained with crystal violet (magnification, 10×/0.25 NA oil). A total of 3 randomly selected fields were examined for cells that had migrated from the top to the bottom chambers. (B-C top) Bar graph represents quantification of migrated cells. Data presented are means ± SD (n = 2; P < .05 for control versus plinabulin). (B-C bottom) Image is representative of 2 experiments with similar results.