Figure 3

Th1 cytokines induce hypoxia-inducible factor-1 mRNA, protein and activity whereas Th2 cytokines have no effect. Cell cultures were exposed to 10 ng/ml cytokine, 1% oxygen, or left untreated for 24 hours. Total RNA was isolated and cDNA generated, and the mRNA level of (a) hypoxia-inducible factor (HIF)-1α and (b) HIF-2α was determined using quantitative PCR. Changes in mRNA levels expressed as fold-change relative to levels in untreated controls (dotted line). Data are mean ± standard error of the mean and were analysed by paired t test of ΔCt values versus control (*P < 0.05, ***P < 0.001). (c) HIF-1 binding to wells pre-coated with human hypoxia-response element oligonucleotides in response to 1% oxygen (hypoxia) and cytokine stimulation, alone or in combination, was measured using nuclear extracts and a TransAm HIF-1 DNA binding kit. DNA binding activity is expressed as the optical density at 450 nm and represents the mean ± standard deviation from three separate representative experiments. Samples were analysed using one-way analysis of variance with Bonferroni's post-hoc test for multiple comparisons versus control (†P < 0.05, ††P < 0.01, †††P < 0.001) or versus hypoxia alone (**P < 0.01, ***P < 0.001). (d) Western blots showing HIF-1α protein levels in response to 1% oxygen, TNFα, IL-4 and cytokines in combination with 1% oxygen for 24 hours. An antibody against α-tubulin was used as a loading control.