Hepatocyte Smad1 or Smad5 single-knockout mice exhibit minimal to no iron loading, whereas knockout of 3 or 4 Smad1/5 alleles causes progressive serum and liver iron overload. (A-B) Serum transferrin saturation (Tf sat; A) and hepatic nonheme iron concentrations (B) of Smad5^fl/fl;Cre⁺ and Smad1^fl/fl;Cre⁺ mice (n = 5-7 per group) compared with their respective littermate Cre⁻ controls. (C-D) Serum transferrin saturation (C) and hepatic nonheme iron concentrations (D) of Smad1^fl/wt;Smad5^fl/fl;Cre⁺, Smad1^fl/fl;Smad5^fl/wt;Cre⁺, and double-knockout Smad1^fl/fl;Smad5^fl/fl;Cre⁺ mice compared with their respective littermate Cre⁻ controls (n = 6-12 per group). Values represent mean ± standard error of the mean. *P < .05; **P < .01; ***P < .001 relative to the respective Cre⁻ controls by Student t test.