Bortezomib represses HIF-1 activity by inhibiting p300 binding to HIF-1α. (A,B) EPO enhancer or VEGF promoter activity in Hep3B cells. Luciferase reporter plasmids (0.5 μg of DNA) containing Epo enhancer (A) or VEGF promoter (B) were cotransfected with 0.5 μg of plasmid cytomegalovirus–β-gal into Hep3B cells. After incubation under normoxic or hypoxic conditions for 16 hours with bortezomib, luciferase activities were measured using a Biocounter M1500 luminometer (Lumac, Bad Wildbad, Germany), and transfection efficiencies were normalized versus β-gal activity. (C) EPO enhancer activity in MM cells. Luciferase reporter plasmids (1 μg) containing EPO enhancer were cotransfected with 1 μg of plasmid cytomegalovirus–β-gal into ARH77 or U266 cells. After a 16-hour normoxic or hypoxic incubation with bortezomib, luciferase activity/β-gal activity was measured. (D) Mammalian 2-hybrid assay of HIF-1α CAD-p300 binding. Hep3B cells were cotransfected with 1 μg of Gal4-luciferase reporter, 1 μg of pGal4-CAD, and 0.5 μg of pVP16-CH1. The cells were incubated under normoxic or hypoxic conditions with bortezomib for 16 hours, and then lysed to determine luciferase activity. All results are presented as relative values versus the normoxic control, and are plotted as means plus or minus SEs of 8 experiments. *P < .05 versus the hypoxic control.