Figure 3.

HIF-1α-target gene expression in the presence of high O₂ consumption and lactate output.A. Transcription levels of five hif-1α target genes; glut1, glut3, pdk1, ldha and hk2, were investigated in virulent (H-V) and attenuated T. annulata-infected (H-A) macrophages. Only ldha levels are lower in H-A macrophages. *Student’s t-test, highest P-value = 0.0074 < 0.05.B. HK2 protein levels remained equivalent in H-V and H-A macrophages. The amounts of p85 were used as a loading control.C. Upper panel, three independent Oroboros measurements show O₂ fluxes are equivalent in both H-A and H-V macrophages and respiration is largely mitochondrial, since upon antimycin (AM) inhibition of complex III O₂ flux is markedly decreased. Lower panel, O₂ consumption over time is the same in H-V and H-A macrophages in a closed Oroboros chamber, which eventually becomes hypoxic. Between each experiment Oroboros chambers were washed 3 times and incubated for at least 5 min with ethanol then rinsed with water.D. Metabolomic analyses highlighting the intracellular metabolite levels stemming from glycolytic activity of H-V and H-A macrophages. H-A macrophages consume more glucose and produce more lactate than H-V macrophages, whereas pyruvate levels are equivalent.