![Figure 3. Combined low doses of NPI-0052 and lenalidomide block migration and tubule formation. (A) For migration assay, MM.1S cells were pretreated with lenalidomide for 12 hours, and then NPI-0052 was added for an additional 6 hours; the cells were more than 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 (original magnification, 10×/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. (Left panel) Image is representative of 2 experiments with similar results. (Right panel) The bar graph represents quantification of migrated cells. Data are mean ± SD (n = 2; P < .05 for control vs NPI-0052 plus lenalidomide–treated cells). (B) HUVECs were cultured in the presence or absence of combined low doses of NPI-0052 plus lenalidomide for 48 hours, and then assessed for in vitro angiogenesis using Matrigel capillary-like tube structure formation assays (original magnification, 4×/0.10 NA oil, media: EBM-2). (Left panel) Image is representative from 3 experiments with similar results. The in vitro angiogenesis is reflected by capillary tube branch formation (dark brown). (Right panel) The bar graph represents quantification of capillary-like tube structure formation in response to indicated agents: Branch points in several random view fields/well were counted, values were averaged, and statistically significant differences were measured using Student t test. (C-D) MM.1S and RPMI-8226 cells were cultured for 48 hours in BMSC-coated or noncoated wells with control media, NPI-0052, lenalidomide, or NPI-0052 plus lenalidomide. Cell proliferation was assessed by the nonradioactive WST-1 colorimetric assay. Data are mean ± SD of 2 independent experiments. Error bars represent SD.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/115/4/10.1182_blood-2009-03-213009/4/zh89990947210003.jpeg?Expires=1723443767&Signature=ONrdY5KFFUJM8hIdeqpDeHefpt5Jc8cIzJcHSVpxT718G42EI86vPPyXo~BPrB7Pxy0QrrEWGv8MClEfMCFuPLk8jmUzrZBKub3E~b2xq8966ogzJsyKGTJF50idKUc7J47PD0jkvicewPtWYTbio5mSvIwGD3zZRbnCS8SYwMvl0MBud~PWxi2kBFFc60~B4n8LViWJzcJviawu0ny-LXC6S1T2F9vypWFWayprKlmiNYNURxMEgQqsPVm1tHzxgXQ6Sspr8mVtTPW6WPYfz1CPQop9dkj0V8wFiUZK6LtV0~6v2A5T5TJ239~rWe2CWgth0WP2qViroKxd1aq1mQ__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Combined low doses of NPI-0052 and lenalidomide block migration and tubule formation. (A) For migration assay, MM.1S cells were pretreated with lenalidomide for 12 hours, and then NPI-0052 was added for an additional 6 hours; the cells were more than 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 (original magnification, 10×/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. (Left panel) Image is representative of 2 experiments with similar results. (Right panel) The bar graph represents quantification of migrated cells. Data are mean ± SD (n = 2; P < .05 for control vs NPI-0052 plus lenalidomide–treated cells). (B) HUVECs were cultured in the presence or absence of combined low doses of NPI-0052 plus lenalidomide for 48 hours, and then assessed for in vitro angiogenesis using Matrigel capillary-like tube structure formation assays (original magnification, 4×/0.10 NA oil, media: EBM-2). (Left panel) Image is representative from 3 experiments with similar results. The in vitro angiogenesis is reflected by capillary tube branch formation (dark brown). (Right panel) The bar graph represents quantification of capillary-like tube structure formation in response to indicated agents: Branch points in several random view fields/well were counted, values were averaged, and statistically significant differences were measured using Student t test. (C-D) MM.1S and RPMI-8226 cells were cultured for 48 hours in BMSC-coated or noncoated wells with control media, NPI-0052, lenalidomide, or NPI-0052 plus lenalidomide. Cell proliferation was assessed by the nonradioactive WST-1 colorimetric assay. Data are mean ± SD of 2 independent experiments. Error bars represent SD.
