Down-regulation of surface wt FPN-c-Myc but not of mutant FPN-c-Myc by hepcidin-25. (A-B) 293T cells were transiently transfected with either c-Myc–tagged wt FPN (A) or c-Myc–tagged C326Y FPN (B) for 2 days with or without 0.5 μM hepcidin-25 (Hep) added. Cells were then stained for surface expression of c-Myc and analyzed by flow cytometry. In the case of wt FPN, hepcidin-25 caused a reduction of detectable cell-surface c-Myc (green line versus red line in panel A), but no reduction of C326Y-c-Myc tag by hepcidin-25 was observed (B). The blue-filled histogram represents the fluorescence of cells stained with an isotype control antibody (anti–CD8-FITC). (C) Quantitation of down-regulation of FPN and FPN mutants by hepcidin-25. Cells were transfected with c-Myc–tagged FPN and FPN mutants with or without added hepcidin-25 and stained for surface c-Myc then analyzed as described in panels A and B. The MFI of the different populations was calculated using CellQuest (BD) software and is displayed as a percentage of the MFI of wt human FPN without added hepcidin-25. Hepcidin-25 reduced surface wt human and murine FPN and Q248H surface expression by at least a half; surface C326Y and Y64N was not affected by hepcidin-25; and N144D and N144H were partially down-regulated. (D) Cells were transfected with c-Myc–tagged FPN and FPN mutants for 24 hours, protein synthesis was inhibited with cycloheximide for 2 hours, and then 0.5 μM hepcidin-25 was added to half the wells for 3 further hours. Cells were then stained for c-Myc expression (green) and cell nuclei were stained by DAPI (blue). Without added hepcidin all FPN variants were localized to plasma membranes (first and third columns from left). In the presence of hepcidin, wt and Q248H FPN were internalized into discrete vesicles, whereas C326Y and Y64N FPN protein remained at the cell surface and both internalized and surface N144D and N144H were observed (second and fourth columns). These results are representative of 3 experiments.