FPN-GFP is rapidly depleted from the phagosomal membrane independently of hepcidin. (A) Schematic depicts the strategy employed to define phagosome “age.” For both time points shown, any bead or phagosome containing a fluorescent bead was excluded from the analysis for FPN-GFP positivity. (B) The distribution of FPN-GFP in relation to phagosomes that can only have existed for a maximum of 5 and 15 minutes is shown. The white arrows point to beads that are demarcated by FPN-GFP, whereas the arrowheads point to beads that have lost the GFP signal. The asterisks in the top row of micrographs highlight 2 beads that can be seen in the DIC but are outside of the fluorescence focal plane and that do show FPN-GFP accumulation. The images shown were acquired from paraformaldehyde-fixed samples and are a z-projection representing the cumulative signal from 5 consecutive z-positions acquired by widefield fluorescence microscopy. Scale bars, 10 μm. (C) RAW macrophages expressing a hepcidin-resistant mutant (C326S) of FPN fused to GFP (FPN(mut)-GFP) was exposed to IgG-opsonized beads. The distribution of FPN(mut)-GFP at the phagosomal membrane was monitored by microscopy at 5, 15, and 60 minutes postaddition of phagocytic targets. The dashed box demarcates the area of the cell presented in the insets. The white arrows point to FPN-positive phagosomes, whereas arrowheads point to FPN-negative phagosomes. Fluorescent micrographs were acquired by widefield microscopy and were taken of fixed cells at the indicated time points. Scale bars, 10 μm. (D) Quantitation of the fraction of FPN-positive phagosomes at the indicated time points is shown for RAW macrophages expressing wild-type FPN or the hepcidin-resistant mutant. The data are the mean ± SEM of 3 independent experiments. Statistical significance was determined by unpaired Student t test at each time point.