Figure 2. HIF-dependent target gene expression is unchanged in PHD3^−/− BMDM.

(A) HIF-1α, HIF-2α, PHD2, and PHD3 protein levels were analyzed after cultivating BMDM for 24 h at 20% or 1% O₂. (B) The expression of HIF-1 target genes, such as PKM2, Pfk, and PDK-1, on mRNA level was analyzed after cultivating BMDM for 24 h at 20% O₂ or 1% O₂ using qRT-PCR. (C) PHD3^−/− [knockout (KO)] and WT BMDM were cultivated for 48 h at 20% O₂ and 1% O₂. Subsequently, cells were lysed and RNA isolated. RNA samples were analyzed in a transcriptome microarray assay. The volcano blots were generated based on the microarray results. (D) PHD3^−/− and WT BMDM were cultured with or without serum for 24 h or stimulated with 100 ng LPS for 12 h. Subsequently, NF-κB activities were analyzed. As a positive control for the NF-κB activity assay, a cell extract of HeLa cells, which were treated with LPS, was used; n = 3; mean ± sd; **P < 0.01, and ***P < 0.001. rel. LU, Relative light units.