Intestine-specific NCOA4 KO mice are protected from systemic iron overload. (A) Schematic depicting the generation of the intestine-specific NCOA4KO (NCOA4^ΔIE) mouse line. (B) Western analysis in the wild-type and NCOA4^ΔIE mice in the duodenum, liver, and spleen. (C) Duodenal NCOA4 gene expression analysis in wild-type vs NCOA4^ΔLiv mice. Characterization of mice treated with vehicle or AV (adenovirus harboring CRISP R-hepcidin guide RNA construct) by assessment for hepatic hepcidin gene expression (D), tissue iron quantification (E), Perl's Prussian blue staining (F), and duodenal NCOA4 gene expression (G). (H) Schematic of AV treatment in NCOA4^ΔIE and wild-type littermates. Hepatic and pancreatic iron (I), serum iron and transferrin (Tf) saturation (J), duodenal enterocyte iron (K), and duodenal FTN H western analyses (L) in the NCOA4^ΔIE and wild-type mice after AV-induced disruption of hepcidin. (M) Schematic of HIF2α/NCOA4 ferritinophagy axis in conditions of intestinal iron hyperabsorption and altered hepcidin-Fpn1 axis, showing active NCOA4-mediated ferritin breakdown facilitates systemic iron loading (left, pink) and how intestine-specific silencing of NCOA4 attenuates it (right, purple). Mice were 4 to 6 weeks of age. Female, brown circles; male, red triangles. All data are mean ± standard error of the mean. *P < .05; **P < .01; P < .001; P < .0001.