Hepcidin regulates endogenous FPN1 expression and intracellular iron levels of fetal liver erythroblasts. (A) Expression of endogenous FPN1 in erythroblasts was decreased after a 24-hour hepcidin treatment. Erythroblasts in proliferation medium were incubated with 1 μg/mL hepcidin for 24 hours, and protein expression was then measured by Western blot analysis for FPN1 and TfR1 in membrane fractions of sample lysates and for L-ferritin in soluble fractions of sample lysates. Ponceau S staining of a specific portion of the gel was used as a loading control. (B) Expression of FPN1 at different time points of incubation with hepcidin during growth in proliferation medium. (C) IRP2 activity was decreased by hepcidin in erythroblasts. Erythroblasts were incubated with 1 μg/mL hepcidin for 24 or 48 hours or with 100μM FeNTA or DFO for 18 hours, and samples were then prepared with or without β-mercaptoethanol to measure IRP activity by electrophoresis mobility shift assay. The density of the bands was analyzed with ImageJ (http://imagej.nih.gov/ij/) and normalized to IRP1 activity after β-mercaptoethanol treatment and statistical comparisons were performed. (D) Western blot analyses showed decreased IRP2 protein levels in samples treated by hepcidin. The cells were treated as described in panel C. (E) Hepcidin increases intracellular iron contents of erythroblasts. Erythroblasts in 1 well of a 6-well plate in proliferation medium were loaded with ⁵⁵Fe by incubation with 5 μCi ⁵⁵Fe (0.185 Bq; 0.2 μg) for 2 hours and were then washed twice and incubated with 1 μg/mL hepcidin for 24 or 48 hours, after which time intracellular ⁵⁵Fe levels were measured with a Liquid Scintillation Counter. (F) Hepcidin increases intracellular iron levels in a dose-dependent manner. Erythroblasts in proliferation medium were loaded with ⁵⁵Fe as described in panel E and were then incubated with hepcidin of indicated concentrations for 24 hours, after which time intracellular ⁵⁵Fe levels were measured with Liquid Scintillation Counter. a.u. indicates arbitrary unit. Experiments in panels A through D were repeated ≥ 3 times, and experiments in E and F were repeated twice with triplicate samples for each time point. *P < .05, **P < .01, and ***P < .001.