Figure 4.

Brusatol increases PHD-mediated HIF-1α degradation by inhibiting hypoxia-induced transition of ferrous iron to its ferric state. (A and B) RKO (A) and HCT116 (B) cells were incubated with or without 100 nM brusatol in the presence and absence of different concentrations of 2,2'-bipyridyl (50-200 μM). After a 1 h incubation, cells were exposed to 20% or 0.5% O₂ for 4 h and then harvested. Whole-cell lysates were analyzed by immunoblotting for the indicated proteins. (C and D) RKO (C) and HCT116 (D) cells were incubated with or without 200 μM 2,2'-bipyridyl or 200 μM FeCl₂ in the presence and absence of 100 nM brusatol. After a 1-h incubation, cells were exposed to 20% or 0.5% O₂ for 4 h and then harvested. Whole-cell lysates were analyzed by immunoblotting for the indicated proteins. (E and F) Left panels and right panels depict HRE-luciferase promoter assay and hydroxylation assay, respectively. RKO (E) and HCT116 (F) cells transfected with p3×HRE-luc (left panels) or pODD-luc (right panels) and pCMV-β-galactosidase were cultured for 16 h, then incubated with or without 200 μM 2,2'-bipyridyl or 200 μM FeCl₂ in the presence and absence of 100 nM brusatol. After a 1-h incubation, cells were exposed to 20% or 0.5% O₂ for 8 h and then harvested. Luciferase activity in whole-cell lysates was normalized to that of β-galactosidase. Data are presented as means ± SD (**P < 0.01, ***P < 0.001, ****P < 0.0001; ANOVA). (G and H) RKO (G) and HCT116 (H) cells were treated with MitoTracker and MitoSOX for 1 h, washed three times with pre-warmed PBS, and exposed to 0.5% O₂ for 4 h. Fluorescence was detected with a Nikon confocal laser-scanning microscope. (I and J) Measurement of intracellular ferrous iron concentrations in RKO (I) and HCT116 (J) cells incubated with or without 100 nM brusatol under normoxia and hypoxia.