Ferritin secretion through EVs is dependent on NCOA4 expression. (A) IMR90SV cells were incubated in the presence or absence of FAC (10 µg/mL) for 48 hours at 37°C. Isolated EVs from cell culture supernatants were collected by the procedures described in “Methods.” (A) Total cell lysates (20 µg of protein per sample) and (C) EVs (20 µg of protein per sample) were analyzed by immunoblotting with anti-CD63, anti-FTH, anti-NCOA4, and anti–β-actin antibodies. (B) Quantification of NCOA4, CD63, and FTH in panel A. (D) Quantification of CD63 and FTH in panel C. Results are mean ± SEM (3 experiments). (E) Schematic illustrating the mechanism of ferritin iron secretion that occurs by EVs upon iron repletion. Iron repletion induces IRP inactivation, which initiates both ferritin and CD63 translation via the dissociation of IRP from the canonical type I IRE in the 5′ UTR of CD63 mRNA. Upon iron repletion, CD63 expression, but not CD81 expression, in EVs is markedly increased, and CD63 is important for ferritin-mediated iron secretion from cells. The ferritin cargo receptor NCOA4 closely associates with CD63⁺ vesicles under iron loading and is necessary for the association of ferritin and CD63 and also ferritin secretion in EVs. It will be necessary to investigate the mechanism of how ferritin is transferred from NCOA4-ferritin complexes⁶⁸ to EVs. In contrast to iron loading, upon iron depletion, ferritin is degraded in lysosomes via the autophagic pathway of ferritinophagy.⁶⁸ Significance was determined by the Student t test. *P < .05; **P < .01; ***P < .001; ****P < .0001.