Effect of hepcidin on WT and mutant Fpn expressed in Xenopus oocytes. (A) First-order rate constants (k) describing ⁵⁵Fe efflux (assayed over 30 minutes) from control oocytes (gray) and oocytes expressing WT Fpn (black) pretreated with 10 µM hepcidin for 0 to 240 minutes (n = 8-12 per group). Two-way analysis of variance (ANOVA) revealed an interaction (P < .001); within Fpn, the 0- and 10-minute time points differed from all other time points (P < .001), and the 30- to 240-minute time points did not differ from one another (P ≥ .35). (B) ⁵⁵Fe efflux in control oocytes and oocytes expressing WT or K8R Fpn that were untreated (−H) or pretreated for 30 minutes with 10 µM hepcidin (+H) (n = 9-12 per group). Two-way ANOVA revealed an interaction (P < .001). Percent inhibition of ⁵⁵Fe efflux by hepcidin did not differ between WT (76% ± 6%) and K8R (71% ± 3%) (means ± standard errors of the means) (P = .47). (C) Live-cell imaging of control oocytes and oocytes expressing WT or K8R Fpn before and after 30 minutes of treatment without hepcidin (−H) or with 10 µM hepcidin (+H) in the same oocyte preparation as used in panel B. Each frame captures portions of 3 oocytes, and the image plane approximately bisects the oocytes. Scale bars, 0.2 mm. Two-way ANOVA of the change in fluorescence intensity (ΔF) over time revealed a greater loss of fluorescence in untreated oocytes (−H) compared with hepcidin-treated (+H) (P = .005) and that ΔF did not differ between WT and K8R (P = .75).