Hepcidin mRNA levels under ex vivo and in vivo hypoxic conditions. (a) Hepcidin mRNA content in HepG2 hepatoma cell lines as determined by Northern blot analysis. HepG2 cells were untreated (20% O₂) or treated with 10, 2, or 0.1% O₂. Twenty micrograms of total liver RNAs were electrophoresed, blotted, and hybridized with both human hepcidin and 18S-labeled probes (as described in Methods). RNAs from HepG2 cells were also hybridized with a human VEGF cDNA probe as a control of the simulation of the vegf gene under hypoxia. (b) Hepcidin mRNA content in liver of mice housed in hypobaric hypoxic chambers for 1, 2, or 4 days, as determined by Northern blot analysis. Twenty micrograms of total liver RNAs were electrophoresed, blotted, and hybridized with both hepcidin and 18S-labeled probes (as described in Methods). Each lane represented different animals. (c) Level of specific hepc1 transcripts in the liver as measured by RT-PCR (as described in Methods). Following PCR, the amplified products (171 bp for hepc1 and 250 bp for β-actin) were separated by electrophoresis on 1.5 % agarose gel.