Loss of PHD2 in cKO P2 mice leads to induction of EPO in kidney and brain. (A) EPO concentration in plasma measured by ELISA in the plasma of WT, cKO P2, cKO P2/H1, and cKO P2/H2 mice (n = 6-22). cKO mice contain on average 6 times more EPO in the circulation than their WT littermates, whereas cKO P2/H1 mice have more than 17 times more EPO. No difference between WT and cKO P2/H2 was found. (B) mRNA levels in total extracts from kidney and brain (n = 5-15) is markedly induced compared with WT but not significantly different between cKO P2 and cKO P2/H1 mice because of the high variation between individual samples (n = 7-11). No difference between WT and cKO P2/H2 could be shown. (C) The renal-derived human cell line (REPC)²⁶  was tested for the expression of EPO and CD68 grown under normoxic and hypoxic conditions via RT-PCR, showing the expected EPO induction and expression of CD68 under both conditions, suggesting a potential link between cre-expression, PHD2 inactivation and subsequent EPO expression in these cell types. (D) Astrocytes isolated from the cerebral cortex of 5 day old pups show a significantly reduced PHD2 content in cKO P2 versus WT mice (n = 3). (E) Typical IHC on brain sections for PHD2 (red) with or without NeuN (green) show a majority of PHD2-negative neurons in the brain of cKO P2 mice (depicted by arrow heads) but not in WT. (F) WT and cKO P2 mice were lethally irradiated and received cKO P2 or WT bone marrow (BM) respectively. HCTs were measured 4 months after transfer. BM from either genotype did not change HCTs in the recipient (n = 6-23). Scale bar in (E) represents 50 μm. All data are mean ± SEM (NS indicates not significant; *P < .05, ***P < .001).