![Figure 4. ONX 0912 blocks migration and tube formation. (A) MM.1S cells were pretreated with 10nM ONX 0912 for 24 hours; the cells were > 90% viable at this time point. The cells were washed and cultured in serum-free medium. After 2 hours of incubation, cells (viability > 90%) were plated on a fibronectin-coated polycarbonate membrane in the upper chamber of Transwell inserts and exposed for 4 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 (×10 original magnification /0.25 numeric aperture [NA] oil). A total of 3 randomly selected fields were examined for cells that had migrated from top to bottom chambers. Image is representative of 3 experiments with similar results. (B) Cells were plated as in panel A, and ONX 0912 effect on migration was assessed in the presence or absence of rVEGF. The bar graph represents quantification of migrated cells. Data presented are means ± SD (n = 3; P = .03 for control versus ONX 0912–treated cells). Error bars represent SD. (C) HUVECs were cultured in the presence or absence of ONX 0912 for 24 hours and then assessed for in vitro angiogenesis using Matrigel capillary-like tube structure formation assays (×4 original magnification/0.10 NA oil, media: endothelium basal medium-2 [EBM-2]). Image is representative from 3 experiments with similar results. The in vitro angiogenesis is reflected by capillary tube branch formation (dark brown). Data represent means ± SD (n = 2; P < .05). (D) In vitro angiogenesis using Matrigel capillary tube formation assay was performed using HUVECs as in panel C. The bar graph represents quantification of capillary-like tube structure formation in response to vehicle alone and ONX 0912–treated cells: Branch points in several random view fields/wells were counted; values were averaged, and statistically significant differences were measured using Student t test. Data presented are means ± SD (n = 3; P = .01 for control versus ONX 0912–treated cells). Error bars represent SD.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/116/23/10.1182_blood-2010-04-276626/4/zh89991061390004.jpeg?Expires=1735498499&Signature=xj~5ZZzFGKcYA0sLhYAY4Gsi8cZbIgONP5BiQSeP55VlxIaiZENwZfEKAmXEBZ4G-WUxxjq6ITukWCkxTlbLTMqfKc1GTl6OSTtdo96ZbG20McdeLG4w4MZ7xdnCaGnhytMYMTPX4d6RiiV6wcYc-VjZ0-t4ciKriLPTO0ZmfMDDUfjwo4FLuo7jriyVmeBR1DMEcxBQQ3bTDG-F9RuYOlD5iZuf3XRt9Tqh9HZOcRA5ybNaCNzAmIpqQ7uZ2QLrIg-2SrvfRzZrBzqZeF~uUj9W2EYsP1ln2JleolIZKD71eLLajZlMITnLWMGc0YGeHFN110akTSZt2r3cx6FhSA__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
ONX 0912 blocks migration and tube formation. (A) MM.1S cells were pretreated with 10nM ONX 0912 for 24 hours; the cells were > 90% viable at this time point. The cells were washed and cultured in serum-free medium. After 2 hours of incubation, cells (viability > 90%) were plated on a fibronectin-coated polycarbonate membrane in the upper chamber of Transwell inserts and exposed for 4 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 (×10 original magnification /0.25 numeric aperture [NA] oil). A total of 3 randomly selected fields were examined for cells that had migrated from top to bottom chambers. Image is representative of 3 experiments with similar results. (B) Cells were plated as in panel A, and ONX 0912 effect on migration was assessed in the presence or absence of rVEGF. The bar graph represents quantification of migrated cells. Data presented are means ± SD (n = 3; P = .03 for control versus ONX 0912–treated cells). Error bars represent SD. (C) HUVECs were cultured in the presence or absence of ONX 0912 for 24 hours and then assessed for in vitro angiogenesis using Matrigel capillary-like tube structure formation assays (×4 original magnification/0.10 NA oil, media: endothelium basal medium-2 [EBM-2]). Image is representative from 3 experiments with similar results. The in vitro angiogenesis is reflected by capillary tube branch formation (dark brown). Data represent means ± SD (n = 2; P < .05). (D) In vitro angiogenesis using Matrigel capillary tube formation assay was performed using HUVECs as in panel C. The bar graph represents quantification of capillary-like tube structure formation in response to vehicle alone and ONX 0912–treated cells: Branch points in several random view fields/wells were counted; values were averaged, and statistically significant differences were measured using Student t test. Data presented are means ± SD (n = 3; P = .01 for control versus ONX 0912–treated cells). Error bars represent SD.
