FIGURE 3

NB-5-MT (2) reduces HIF-1α stability through pVHL binding. A, The dose dependence of 2 on HIF-1α expression. Under hypoxia, HCT116, HeLa, and MDA-MB-231 cells were treated with various concentrations (0.125-1.0 mM) of 2 for 4 hr HIF-1α expression was examined by western blot analysis. B, The effect of 2 on the stability of endogenous HIF-1α. Under hypoxia, HCT116 cells were incubated with or without 1.0 mM of 2 in the presence of the proteasome inhibitor MG132 for 4 hr (C) The effect of 2 on the stability of exogenous HIF-1α and the expression of VEGF. Empty vector (EV) or HIF-1α expression plasmid was transiently transfected in HCT116 cells. After 24 h, HCT116 cells were incubated with or without 1.0 mM of 2 in the presence of the proteasome inhibitor MG132 for 4 hr (D) The effect of 2 on the binding between HIF-1α and pVHL. Under hypoxia, HCT116 cells were incubated with or without 1.0 mM of 2 in the presence of the proteasome inhibitor MG132 for 4 hr, and then 500 μg of total protein was immunoprecipitated with either anti-IgG or anti-VHL antibodies. Western blotting was performed with anti-HIF-1α and anti-pVHL antibodies