Combined low doses of NPI-0052 and bortezomib block migration and tubule formation. (A) Growth factor–deprived MM.1S cells were either pretreated with indicated concentrations of NPI-0052, bortezomib, or a combination of NPI-0052 and bortezomib. Cells were then plated on a fibronectin-coated polycarbonate membrane in a modified Boyden chamber and exposed for 8 hours to serum containing medium in the lower chamber. The migrated cells on 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 top to bottom chambers. Image is representative of 2 experiments with similar results. (B) MM.1S cells were treated with the indicated concentrations of NPI-0052, bortezomib, or NPI-0052 plus bortezomib for 4 hours (viability greater than 95%), washed, and then treated for 24 hours with rVEGF (10 ng/mL), followed by analysis in a Transwell migration assay. The bar graph represents quantification of migrated cells. Data represents means plus or minus SD of 2 independent experiments (P < .05 for samples treated with rVEGF alone versus NPI-0052 plus bortezomib–treated cells). (C) HUVECs were treated with the indicated concentrations of NPI-0052, bortezomib, or combined NPI-0052 plus bortezomib and assessed for in vitro angiogenesis using Matrigel capillary-like tube structure formation assays (magnification: 4×/0.10 NA oil media: EBM-2). Data represent means plus or minus SD (n = 3; P < .05). The in vitro angiogenesis is reflected by capillary tube branch formation (dark brown). Image is representative of 3 experiments with similar results. (D) HUVECs were treated with the indicated concentrations of NPI-0052, bortezomib, combined NPI-0052 plus bortezomib, or lenalidomide (5 μM) and assessed for in vitro angiogenesis using Matrigel capillary-like tube structure formation assays as in panel C. The bar graph represents quantification of in 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 significance differences were measured using the Student t test. Error bars represent standard deviation.