Combined low doses of FK866 and bortezomib overcome the survival advantage conferred by the BM microenvironment and inhibit in vitro capillary-like tube formation. (A) MM1S cells were cultured for 72 hours in BMSC-coated or -uncoated wells with control media, FK866, bortezomib, or FK866 plus bortezomib. Cell proliferation was assessed by [³H]thymidine incorporation assay. Data are mean ± SD of triplicate samples. Error bars represent SD. (B) MM1S cells were treated with FK866, bortezomib, or their combination in the presence or absence of rhIL-6 (10 ng/mL) or rhIGF-1 (100 mg/mL) for 72 hours; DNA synthesis was then determined by [³H]thymidine uptake. The results are mean ± SD of triplicate samples. (C) HUVEC were treated with FK866 (10 nM), bortezomib (2.5 nM), or their combination for 8 hours and then assessed for in vitro angiogenesis using Matrigel capillary-like tube structure formation assay. Endothelial cell tube formation was analyzed by microscopy (magnification: ×40/ DIC NA 0.75). Image is representative of 3 experiments with similar results (left). Bar graph represents quantification of tube formation in left panel in response to indicated stimuli: branch points in several random views fields/well were counted, values were averaged, and statistical significance of differences was measured using the Student t test (right). Error bars represent SD. CPM, [³H]thymidine incorporation.