Hypoxic response is fully functional in vitamin C–depleted Gulo^−/− mice.Gulo^−/− male mice received a diet with (+Asc) or without (−Asc) ascorbate for 5 weeks. (A) Transcript levels of HIF target genes (Bnip3, Ca9, Glut1, Pdk1, Phd2, Phd2, Phd3, and Ndrg1) as well as genes involved in antioxidative defense (Sod1, Sod2, and Glrx), the ascorbate transporters Svct1 and Svct2, or oxygen-independent genes (Ednrb, Mmp3, and Phd1) were quantified by RT-qPCR in brain, kidney, lung, liver, and heart. Values are expressed relative to S12 mRNA levels and visualized in a heatmap (Genepattern; Broad Institute). Lowest and highest mRNA levels of each gene were arbitrarily defined as −3 (dark blue) and +3 (dark red), respectively. (B) Heatmap of gene expression changes following a vitamin C–deficient diet. Log₂ (−Asc/+Asc) ratios revealed that the majority of HIF target genes remained either unchanged or showed a slightly reduced expression pattern. Data ranged from −1.75 (dark blue) and +1.79 (dark red), respectively. (C) Scheme depicting the experimental setup for hypoxic experiments with vitamin C–depleted Gulo^−/− animals (top panel). Relative gain of body weight of Gulo^−/− mice (n = 11 animals per group) after ascorbate withdrawal (−Asc) or ascorbate supplementation (+Asc) for 36 days (left panel). Ascorbate levels in the plasma of Gulo^−/− mice (n = 6 animals per group) after 5 weeks of ascorbate withdrawal compared with mice kept on an ascorbate-supplemented diet (right panel). (D) Epo mRNA (left panel) and circulating EPO protein (right panel) levels in Gulo^−/− mice maintained on a diet with (+Asc) or without (−Asc) for 5 weeks followed by exposure to 8% or 21% oxygen for 24 hours. Data represent mean values ± SEM derived from at least 5 animals per group; n.s., indicates not significant.