Effects of hepcidin on FPN and mutant FPN-mediated changes in iron accumulation and iron release. (A-B) Effect of hepcidin-25 on iron accumulation by FPN and FPN mutant-expressing cells. Cells were transfected with FPN and FPN mutants and cultured with 40 μg/mL ⁵⁹Fe-Tf with or without 0.5 μM hepcidin-25 added for 2 days, and then cellular ⁵⁹Fe accumulation per million cells was determined. Hepcidin did not alter ⁵⁹Fe accumulation by control CD8-transfected cells. Expression of FPN and all FPN variants reduced iron accumulation, consistent with enhanced iron export by transfected cells. Hepcidin-25 reversed the effect of wt and Q248H FPN but did not inhibit C326Y or Y64N FPN and only marginally increased iron accumulation by cells expressing N144D and N144H FPN. Each point represents the iron accumulation by one aliquot of a million cells. Red bar shows mean ⁵⁹Fe accumulation (± 95% CI). ^*Significance compared with CD8 control (P < .001 by Student t test). (C) Hepcidin-25 inhibits iron release from cells expressing wt FPN but not C326Y FPN. 293T cells were preloaded with ⁵⁹Fe, then transfected for 15 hours, washed, and recultured in serum-free medium and the percentage ⁵⁹Fe released (of starting) was measured over 32 hours. Hepcidin does not affect “background” release from control 293T cells transfected with GFP (or CD8, not shown). Data shown are representative of 2 or more experiments for each mutant.