ERFE overexpression exacerbates iron overload in thalassemic mice. Mice generated from Th3 × E(M) breedings were analyzed at 16 weeks of age. Males are depicted as closed circles and females as open circles. (A-E) Nonheme iron concentrations were assessed in (A) serum, (B) liver, (C) kidney, and (D) spleen: (E) total splenic nonheme iron, (F) iron visualized in formalin-fixed paraffin-embedded sections sections of the kidney (top), liver (middle) and spleen (bottom) from male 16-week-old mice using diaminobenzidine-enhanced Perls’ stain (kidney and liver) and Perls’ stain (spleen). Original magnification ×10 (F). Scale bars represent 200 μm (F). (A-F) P values were assessed by two-tailed unpaired t test (∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001) between the Th3 and T-E(M) groups only.