Erythrophagocytosis activates autophagy in BMDMs, and autophagy deficiency in BMDMs abrogates ferroportin degradation. (A) Expression kinetics in BMDMs treated with oRBCs. Atg5^−/− and WT BMDMs were incubated with oRBCs (oRBCs to cells, 5:1) for the indicated times. Western blot analysis of Hif2α, p62, and LC3 from total protein extracts and Fpn from membrane extracts, and β-actin as control, in BMDMs from LysM-Atg5^−/− and WT mice incubated with oRBCs (oRBCs to cells, 5:1) for the indicated times. After treatment with oRBCs, protein was extracted from BMDMs for western blot analysis (representative analysis of n = 3 independent experiments is shown; quantitative analysis of the western blots is given in supplemental Table 4). (B) Kinetics of Hmox1, Fpn, and Hamp1 mRNA expression in BMDMs treated with oRBCs. Representative mRNA fold changes (relative to Gapdh) expressed as −ΔΔCt values (n = 3 replicates). Data are presented as means and standard errors. (C) Iron loading and ferroportin localization in BMDMs from LysM-Atg5^−/− and WT mice treated with oRBCs. (Ci) Representative images of Perl’s/DAB iron–stained BMDMs (NanoZoom scan imaging; original magnification, 40×). BMDMs cultured on slides incubated for the indicated times after treatment with oRBCs (oRBCs to cells, 5:1) were fixed with paraformaldehyde for immunostaining or with methanol for Perl’s staining followed by DAB enhancement. (Cii) Detection of ferroportin by immunofluorescence in BMDMs from LysM-Atg5^−/− and WT mice. Images are composites of ferroportin (red) and 4′,6-diamidino-2-phenylindole (DAPI)–stained nuclei (blue) (Leica fluorescence microcopy imaging; original magnification, 100×; representative images shown are from n = 4 independent experiments). (D) Immunofluorescence colocalization of ferroportin in BMDMs from WT mice treated with oRBCs. WT BMDMs were double labeled with a rabbit anti-Fpn antibody and Alexa Fluor 488–conjugated secondary donkey anti-rabbit immunoglobulin G (IgG; red), followed by rabbit anti-Atg5 antibody and TRITC-conjugated goat secondary anti-rabbit IgG (green). Cell nuclei were stained with DAPI (blue). Erythrophagocytosis resulted in colocalization of ferroportin and Atg5 in erythrophagocytic BMDMs at 8 to 16 hours (Leica fluorescence microcopy imaging; original magnification, 40×; representative images shown are from n = 2 independent experiments). (E) Immunofluorescence colocalization of ferroportin and hepcidin in BMDMs from LysM-Atg5^−/− and WT mice treated with oRBCs. WT and Atg5^−/− BMDMs were double labeled with a rabbit antiferroportin antibody and Alexa Fluor 488–conjugated secondary donkey anti-rabbit IgG (red), followed by rabbit antihepcidin antibody⁵⁸ and TRITC-conjugated secondary goat anti-rabbit IgG (green). Cell nuclei were stained with DAPI (blue). Erythrophagocytosis resulted in colocalization of ferroportin and hepcidin in WT cells at 12 hours and Atg5^−/− cells at 16 hours (Leica fluorescence microcopy imaging; original magnification, 40×; representative images shown are from n = 2 independent experiments). *P < .05, **P < .01, ***P < .001, ****P < 10⁻⁴.