Figure 2. HIF1α, but not HIF2α, activation induces aerobic glycolysis and decreases mitochondrial activity in fibroblasts. (A) MitoTracker staining (red) was used to visualize mitochondrial activity. Nuclei were stained with Dapi (blue). MitoTracker accumulates in functional mitochondria with an active membrane potential, thus correlating with mitochondrial activity. Note that fibroblasts harboring activated HIF1α show reduced MitoTracker staining, consistent with a shift toward aerobic glycolysis. Conversely, fibroblasts harboring activated HIF2α show similar MitoTracker staining as the empty vector control cells. (B) Empty vector control, HIF1α- or HIF2α-activated fibroblasts were grown for 48 h in media with 2% FBS. Then, the media was collected and subjected to a biochemical analysis to determine the concentration of lactate. The graph shows that HIF1α-activated fibroblasts secrete 2-fold more lactate than empty vector control cells. Conversely, HIF2α overexpressing fibroblasts show a 1.5-fold reduction in lactate production, as compared with control cells. p values, Student t-test. (C) Immunoblotting was performed on control, HIF1α- and HIF2α-fibroblasts with antibodies against MCT4 (the transporter for lactate efflux) and MCT1 (the transporter for lactate uptake). HIF1α-activated fibroblasts show MCT4 upregulation, and MCT1 downregulation, relative to control cells, consistent with increased lactate production. Conversely, HIF2α-activated fibroblasts show robust expression of both MCT1 and MCT4, suggesting that the lactate flux is not unidirectional in HIF2α-activated cells. Equal loading was assessed by immunoblotting with β-actin.