Foxo1 ablation impairs the response of hepcidin signaling to iron. (A) Nuclear Foxo1, Smad1, Smad4, and Smad5 were measured in the primary hepatocytes from 8-week-old male Foxo1^LKO and Foxo1^flox/flox mice receiving normal or a 2-week HID (left). Quantification of the nuclear Foxo1, Smad1, Smad4, and Smad5 proteins (right). (B) Primary hepatocytes from 8-week-old male Foxo1^LKO and Foxo1^flox/flox mice were treated with holo-Tf and/or BMP6; hepcidin (Hamp1) mRNA expression was measured by qPCR at indicated time point. (C) Primary hepatocytes from 8-week-old male Foxo1^LKO and Foxo1^flox/flox mice were treated with holo-Tf and/or BMP6 for 7 hours; the nuclear translocation of Foxo1, Smad1, Smad4 and Smad5 were measured by nucleocytoplasmic separation (left) and quantified (right). The concentration of holo-Tf was 30 μM and BMP6 was 20 ng/mL. Results from qPCR were obtained from 3 biological replicates. Quantification of western blot was obtained from 3 independent experiments. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001. The P value was calculated by the 1-way ANOVA with the Sidak multiple comparison test. (B) Multiple comparison test was performed in groups with the same treatment duration. (C) Multiple comparison test was performed in groups with the same treatment condition.