Hamp1 expression in LysM-Atg5−/− mice. (Ai) Liver Hamp1, Id1, and Fga mRNA levels (relative to Gapdh) expressed as −ΔCt values and correlation of Hamp1 and Id1 mRNA levels (n = 8 per group). (Aii) Ratio of liver Hamp1 mRNA levels to hepatic iron content. (Aiii) Detection of hepcidin by immunofluorescence in liver tissue from LysM-Atg5−/− and WT mice. Representative images are composites from hepcidin (red) and 4′,6-diamidino-2-phenylindole (DAPI)–stained nuclei (blue) (n = 4 animals per group; Leica fluorescence microcopy imaging; acquisition time, 3.000 ms; original magnification, 10×; scale bars, 50 μm). (B) Dot blotting of serum hepcidin from LysM-Atg5−/− and WT mice; 5 μL of serum samples were spotted. Three samples per genotype and controls of WT mice stimulated with lipopolysaccharide (LPS; 5 μg/25 g) 24 hours before necropsy are shown (control dot blots of 10% milk and serum from WT mice fed iron-replete, iron-deficient, and iron-rich diets are shown in supplemental Figure 4D; densitometric analysis was performed on n = 6 samples per group, and P values are given). (Ci) Spleen Hamp1, Id1, and Fga mRNA levels (relative to Gapdh) expressed as −ΔCt values (values are the mean of n = 6 experiments). Data are presented as means and standard errors (n = 6 per group). (Cii) Detection of hepcidin by immunofluorescence in spleen from LysM-Atg5−/− and WT mice. Representative images are composites from hepcidin (red) and DAPI-stained nuclei (blue) (n = 4 animals per group; Leica fluorescence microcopy imaging; acquisition time, 100 ms; original magnification, 10×; scale bars, 50 μm). (D) Detection of hepcidin by immunofluorescence in duodenum from LysM-Atg5−/− and WT mice. Representative images are composites from hepcidin (red) and DAPI-stained nuclei (blue) (n = 3 animals per group; Zeiss inverted microscopy imaging; original magnification, 10×; scale bars, 20 μm). Hepcidin protein was detected using a specific rabbit anti–hepcicin-25 antibody.58 *P < .05, **P < .01, ***P < .001. RP, red pulp; WP, white pulp.