Single dose of RO-68 (60 mg/kg) strongly inhibited liver hepcidin and spleen iron in 6 hours. Mice (8-9 weeks old) were treated SC with RO-68 (60 mg/kg) in a single dose and euthanized after 1-3-6-12-16 hours. (A) Hepcidin mRNA was quantified by qRT-PCR in relationship to Hprt1 mRNA. (B) Serum hepcidin was measured by surface enhanced laser desorption ionization–time of flight mass spectrometry after 6 hours of treatment. (C) Spleen iron was measured spectrophotometrically. (D) pSMAD1, pSMAD15, and pSMAD18 were evaluated by western blotting analysis. Actin was used as calibrator. Data are representative of 4 mice/point and are expressed with box plot histograms. Hepcidin level is expressed as percentage of untreated mice (100%)