Role of HIF-1α and -2α in the hypoxic induction of VEGFA and CXCL8: insights from siRNA knockdown studies and use of macrophages bearing a deletion in the HIF-1α gene. (A) Immunoblots of HIF-1α or HIF-2 α in MDM lysates after their exposure to normoxia (20.9% O2; N) or hypoxia (0.1% O2; H) for 18 hours, or hypoxia for 18 hours after exposure to siRNA for HIF-1α (1α), HIF-2 (2α), both HIFs-1α and 2α together (1α + 2α), or a scrambled control (Scr). Loading controls were β-actin. Vertical lines have been inserted to indicate repositioned lanes from the same gel. Below each gel picture is the densitometric analysis of HIF expression relative to its β-actin loading control. (B-C) Effects of HIF-1α and -2α knockdown on the hypoxic induction of VEGFA (B) and CXCL8 (IL-8; C) mRNA and protein. In the case of VEGF, gene expression was also assayed in normoxic and hypoxic BMDMs from mice bearing a myeloid cell-specific knockout of the HIF-1 α gene (HIF-1α−/−) in vitro by quantitative RT-PCR (B right panel). It was not possible to do this for CXCL8, as this gene is not expressed in mice. Pooled data from 6 replicate experiments are shown. *P < .05 compared with corresponding normoxic group. ∧P < .05 compared with the scr siRNA/hypoxia group. +P < .05 compared with macrophages from wild-type mice exposed to hypoxia.
