Bortezomib inhibits the hypoxic induction of EPO and VEGF in both MM and non-MM cells. (A) Effects of bortezomib on the hypoxic induction of EPO or VEGF mRNA. Hep3B, ARH77, and U266 cells were incubated under normoxic (N) or hypoxic (H) conditions for 16 hours in the presence of various concentrations of bortezomib (Bort). Total RNAs were extracted, and EPO, VEGF, and β-actin mRNA were analyzed by semiquantitative RT-PCR. (B) Effects of bortezomib on expressions of HIF-1 subunits. Under normoxic or hypoxic conditions for 16 hours, Hep3B cells were lysed in a denaturing SDS sample buffer, and HIF-1α, ARNT, and β-tubulin proteins were analyzed by Western blotting. The data shown are representative of 3 separate experiments. (C) Chemosensitivity to bortezomib. Cell viabilities were measured using an MTT-labeling method, as described in “Chemosensitivity assays.” Hep3B (□), ARH77 (○), and U266 (△) cells were treated with various concentrations of bortezomib for 48 hours. Points represent the means plus or minus SD of 6 experiments. The iHR box indicates the concentration range showing inhibited hypoxic responses.