Figure 4

BRU promotes degradation of HIF-1α protein in hypoxic conditions.

(A) HCT116 cells were cultured under normoxia or hypoxia (1% O₂) in the presence or absence of 10 μM MG132, with or without 60 nM BRU, for 4 h and the quantity of HIF-1α protein was analyzed by Western blot. (B) HCT116 cells were cultured under normoxia or 200 μM CoCl₂-induced mimic hypoxia in the presence or absence of 10 μM MG132, with or without 60 nM BRU, for 4 h and the quantity of HIF-1α protein was analyzed by Western blot. (C) and (D) HCT116 cells were firstly incubated with MG132 in normoxia. Then, CHX (50 μg/ml)-containing fresh medium was added into cells, and the cells were further incubated in hypoxia for different time in the presence or absence of 60 nM BRU. At each time point, cells were harvested and the quantity of HIF-1α protein was analyzed by Western blot. (E) and (F) HCT116 cells were firstly incubated with MG132 in normoxia. Then, CHX (50 μg/ml)-containing fresh medium was added into cells, and the cells were further incubated in CoCl₂-induced mimic hypoxia for different time in the presence or absence of 60 nM BRU. At each time point, cells were harvested and the quantity of HIF-1α protein was analyzed by Western blot. (G) The relative change in the HIF-1α protein levels at each time point described in (C) and (D). Data was presented as means ± S.D. (n = 3). ^**p < 0.01 and ^*p < 0.01 versus the hypoxia alone group. (H) The relative change in the HIF-1α protein levels described in (E) and (F). Data was presented as means ± S.D. (n = 3). ^**p < 0.01 and ^*p < 0.01 versus the mimic hypoxia alone group.