Monoferric strains produce a hemochromatosis phenotype that is more pronounced upon loss of iron binding in the N lobe. (A) Hepatic iron concentrations were significantly higher in the Tf^N-bl/N-bl strain than in the Tf^C-bl/C-bl strain and higher in both mutant strains than in WT (n = 9-10 mice per sex). (B) Enhanced Perls’ staining showed higher iron in the Tf^N-bl/N-bl and Tf^C-bl/C-bl mice compared with WT. (C) Splenic iron concentrations were significantly lower in both the Tf^N-bl/N-bl and the Tf^C-bl/C-bl mice than in WT mice (n = 10-12 mice per sex). (D) Enhanced Perls’ staining confirmed decreased splenic iron in the Tf^N-bl/N-bl and Tf^C-bl/C-bl mice compared with WT littermates. (E) Serum hepcidin levels were decreased in the Tf^N-bl/N-bl mice compared with WT (n = 11 WT females and n = 4-7 females for each mutant strain). (F) Upon normalization to mean liver iron concentrations (LICs) for each strain, serum hepcidin values were significantly lower in both Tf-mutant strains compared with WT and significantly lower in the Tf^N-bl/N-bl strain compared with the Tf^C-bl/C-bl strain. Quantitative PCR data, presented as the fold change in expression relative to β-actin, demonstrated no significant differences in Bmp6 mRNA levels (n = 8-10 mice per sex) (G) or Bmp2 mRNA expression (n = 3-4 mice per sex) (H). Data are presented as Tukey box and whisker plots. Statistical significance was analyzed by Kruskal-Wallis test or 1-way ANOVA. *P ≤ .05, **P≤ .01, ***P ≤ .001, ****P ≤ .0001.