Rnf217^−/− macrophages have FPN accumulation and impaired iron homeostasis. (A) Hepatic and splenic nonheme iron concentration was measured in 2- and 5-month-old Rnf217^flox/flox and Rnf217^Lysm/Lysm mice (n = 6-7 mice per group). (B) Spleen sections were prepared from 2- and 5-month-old Rnf217^flox/flox and Rnf217^Lysm/Lysm mice and immunostained for FPN (n = 4 mice per group). (C) Intracellular iron was measured in Calcein-AM‒loaded BMDMs treated as indicated (n = 3 mice per group). The indicated BMDMs were pretreated with FAC (100 µM, 12 hours) and then treated with CHX (75 µM) for the indicated times; (D) FPN was then measured using western blot analysis and (E) quantified (n = 3 mice per group). (F) Rnf217^flox/flox and Rnf217^Lysm/Lysm mice were fed a high-iron diet for 6 weeks followed by a low-iron diet for 5 weeks, after which hepatic and splenic nonheme iron concentration were measured (n = 6-8 mice per group). (G) Western blot analysis and (H) quantification of FPN measured in spleen samples prepared from Rnf217^flox/flox and Rnf217^Lysm/Lysm mice injected with either saline or hepcidin (n = 5-6 mice per group). (I) Serum iron was measured in Rnf217^flox/flox and Rnf217^Lysm/Lysm mice injected with either saline or hepcidin (n = 4-5 mice per group). (J) Serum iron was measured in Rnf217^flox/flox and Rnf217^Lysm/Lysm mice injected with either saline or LPS (5 µg/g body weight) (n = 3-5 mice per group). (K) Western blot analysis and (L) quantification of FPN measured in spleen samples prepared from Rnf217^flox/flox and Rnf217^Lysm/Lysm mice injected with either saline or LPS (n = 4-6 mice per group). Scale bars, 100 μm (B). *P < .05; **P < .01 (Student t test); in panels E, H-J, and L, groups labeled without a common letter were significantly different (P < .05; 1-way ANOVA).