Chronic treatment with RO-82 and RO-68 (20-60 mg/kg daily for 1 week) inhibited liver hepcidin and spleen iron. Mice (8-9 weeks old) were treated with RO-82 and RO-68 (20-60 mg/kg daily for 1 week) and euthanized. Hepcidin (A) and Id1(B) mRNA were quantified by qRT-PCR in relationship to Hprt1 mRNA. Data are representative of 4 mice/point and are expressed with box plot histograms. Hepcidin and Id1 level are expressed as percentage of untreated mice (100%). pSMAD1/5/8 and SMAD1 were evaluated in untreated and treated (60 mg/kg RO-82 and RO-68) mice by western blotting (C). Serum hepcidin was measured by surface enhanced laser desorption ionization–time of flight mass spectrometry (D), and spleen iron was measured spectrophotometrically (E).