Intestinal NCOA4 is a HIF2α target gene. (A) NCOA4 gene expression analysis by quantitative polymerase chain reaction (qPCR) in mouse tissues; data were normalized to β-actin values, followed by fold change comparison with splenic NCOA4 expression. Wild-type mice were treated with 350- or <5-ppm iron diets for 2 weeks, followed by duodenal NCOA4 gene expression (B) and western blot analysis (C). (D) NCOA4 gene expression analysis in undifferentiated (Undiff.) and differentiated (Diff.) human intestinal organoids treated with deferoxamine (DFO), 100 μM or 1% O₂ for 16 hours. Wild-type (HIF2α^F/F) and intestine-specific HIF2α (HIF2α^ΔIE) KO mice were treated with 350- or <5-ppm iron diets for 2 weeks, followed by duodenal NCOA4 gene expression (E) and western analysis (F). Duodenal NCOA4 gene expression in vehicle (phosphate-buffered saline) or FG4592-treated mice (G), and wild-type (Vhl^F/F) or intestine-specific Vhl KO (Vhl^ΔIE) mice (H). (I) Duodenal NCOA4, FTN H, IRP2, and HIF2α western blot analysis in Vhl^F/F and Vhl^ΔIE mice. (J) Duodenal NCOA4 and FTN H western analysis in Vhl^F/F and Vhl^ΔIE mice from lysosomal fraction. Duodenal NCOA4 gene expression analysis (K), and duodenal NCOA4 and FTN H western analysis (L) in wild-type (Vhl/HIF2α^F/F) and intestine-specific Vhl-HIF2α double-KO (Vhl/HIF2α^ΔIE) mice. (M) NCOA4 promoter analysis by luciferase assay in HCT116 and Caco-2 cells cotransfected with empty vector (EV), HIF-1α, or HIF-2α. (N) Chromatin immunoprecipitation (IP) assay in the Vhl^ΔIE duodenal epithelial cells relative to normal anti-rabbit IgG. Mice were 4 to 6 weeks old. Female (brown circles); male (red triangles). All data are mean ± standard error of the mean. *P < .05; **P < .01; ***P < .001; ****P < .0001.