HBP inhibits hepcidin-induced ferroportin internalization. (A-D) Immunofluorescence micrographs of FPN-EGFP–expressing COS-1 cells. Cells were maintained in Dulbecco's modified Eagle high-glucose medium (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS; Capricorn Scientific GmbH) and incubated in serum-free medium in the presence of cycloheximide (100 μg/mL) for 2 hours before the experiment. (A) FPN-EGFP–expressing COS-1 cells were incubated in serum-free medium with no peptides. (B) FPN-EGFP–expressing COS-1 cells were incubated with hepcidin (0.5μM). (C) The cells were incubated with hepcidin (0.5μM) plus HBP (5.0μM). (D) The cells were incubated with hepcidin (0.5μM) plus a control peptide (5.0μM). Peptides and hepcidin were simultaneously added to the cell media. FPN-EGFP was internalized, and the punctate intracellular vesicles containing FPN-EGFP were observed. The white arrowheads denote the representative intracellular vesicles. (E) Changes in the number of the FPN-EGFP–containing vesicles per cell. The bars represent means ± standard error (SE; n = 3). ∗P < .05. Cont., control peptide.