PR65 prevented iron loading in iron-depleted hepcidin knockout mice. All mice were placed on an iron-deficient diet (4 ppm iron) from ages 5 to 6 weeks until 12 weeks. The “baseline” group (n = 7) was examined at 12 weeks of age (white bars). The rest of the mice were fed an iron-loading diet (300 ppm) for 2 more weeks while receiving daily subcutaneous injections of solvent (gray bars, n = 6) or PR65 at 20, 50, or 100 nmol per day (black bars, n = 4 per dose). The mice were analyzed 24 hours after the last injection. Compared with solvent, PR65 injections resulted in: (A) iron retention in the spleen, (B) a dose-dependent decrease in serum iron, (C) a corresponding dose-dependent decrease in Hb levels, (D) a decrease in heart iron at higher doses, and (E) decreased liver iron. Liver iron content in PR65-injected mice did not significantly differ from that in the baseline group of mice, indicating that little to no new iron was absorbed or deposited in the liver during the 2-week treatment. Graphs show means and standard deviations. Student t test was used to compare the mean of each condition to that of solvent treatment (P value over bars). In panel E, mean of each condition was also compared with the baseline (P values at lines over bars).