Figure 4. BACH1 expression under normoxia and hypoxia is HIF1α dependent.

(A) RT-qPCR of HIF1A and HIF2A in spheroids incubated for 7 days with antioxidants under normoxia (n = 3 experiments). (B) Top: HIF1α levels in spheroids incubated with VitC by Western blotting. Middle: HIF1α levels by densitometry (n = 3 experiments). Bottom: HIF2α protein levels by Western blotting. (C) RT-qPCR of BACH1 expression in spheroids under normoxia (21% O₂) and hypoxia (1% O₂) (n = 3 experiments), BACH1 protein levels by Western blotting, and BACH1 levels by densitometry (n = 3 experiments. (D) Left top: BACH1 protein levels by Western blotting in spheroids incubated for 16 hours with prolyl hydroxylase inhibitors. Right top: BACH1 levels by densitometry (n = 3 experiments). Left bottom: HIF1α protein levels by Western blotting. Right bottom: HIF1α levels by densitometry (n = 3 experiments). (E) Top: RT-qPCR of BACH1 expression in HIF1α-overexpressing (HIF1A^OE) and control (HIF1A^WT) spheroids under normoxia (n = 3 experiments). Middle: BACH1 protein levels by Western blotting. Bottom: BACH1 levels by densitometry (n = 4 experiments). (F) Experiments similar to those in E using HIF2α-overexpressing (HIF2A^OE) and control (HIF2A^WT) spheroids (n = 6 experiments). Data indicate the mean ± SEM. P values were determined by 2-tailed, unpaired Student’s t test (A, C, E, and F) and 1-way ANOVA with Tukey’s post hoc test for multiple comparisons (B and D).