Iron deficiency is independent of hepcidin and other known regulatory mechanisms in cre/+ mice. (A) Liver Hamp (hepcidin) mRNA expression in control (+/+) and cre/+ mice (∗P < .05), and (B) in cre/+ mice after everolimus treatment (∗∗P < .01). (C-D) Hepcidin concentration measured in the serum (C) and in duodenum lysates (D) of control (+/+) and cre/+ mice via enzyme-linked immunosorbent assay. (E) Representative western blot analysis of Cp (ceruloplasmin [ferroxidase], 122 kDa), FPN1 (ferroportin-1 [cellular iron exporter], 56 kDa), iron regulatory protein/acotinase1 (iron regulatory protein [IRP1/Aco1], 98 kDa), transferrin (75 kDa), and the housekeeping protein α-tubulin (55 kDa) in the liver and spleen lysates from control (+/+) and cre/+ mice. (F) Representative western blot of Cp (122 kDa), FPN1 (56 kDa), and the housekeeping protein α-tubulin (55 kDa) in duodenum lysates from control (+/+) and cre/+ mice. (G) Representative image of immunofluorescent staining for FPN1 and F4/80 (macrophage marker) on mouse duodenum cryosections obtained from control (+/+) and cre/+ mice. Scale bar, 50 μm. Quantification of FPN1 mean fluorescence intensity in LP of duodenum. (H) Representative image of immunohistochemistry staining for hephaestin on mouse duodenum FFPE sections obtained from +/+ and cre/+ mice. Scale bar, 100 μm. (I) Representative western blot analysis of iron regulatory protein IRP1/Aco1 (iron regulatory protein/acotinase 1, 98 kDa) and the housekeeping protein α-tubulin (55 kDa) in duodenum lysates from control (+/+) and cre/+ mice. (J) Volcano-plot of normalized gene expression profiles in duodenum epithelial cells from control (+/+) and cre/+ mice.