**2.2 Assembly and cysteine oxidation of HepcH-FTH heteropolymers**

To enhance the assembling of HepcH as an heteropolymer molecule, it was mixed, with the molar ratio hepcidin/ferritin of 1:5, in presence of denatured FTH in 6 M GdnHCl pH 7. The mixture was then diluted at least 10-fold into 0,1 M sodium phosphate pH 7,4, in the presence of 5 mM beta-mercaptoethanol (3 mM DTT and 1 mM EDTA), and incubated with stirring for 18 h at 4°C, for the renaturation of the heteropolymer. The diluted solution was then clarified by centrifugation, at 4000RPM for 15 min, at least twice to separate the soluble fraction from the insoluble cellular debris. A 10-fold concentration was performed with a 100-KDa molecular weight cutoff membrane. The renatured HepcH-FTH heteropolymer was then purified using a gel filtration on a Sepharose 6B column and analyzed on native gel 8%. A slightly modified protocol of HepcH-FTH renaturation, using different molar ratios of hepcidin/ferritin, was described by Boumaiza et al. [2]. Both protocols showed to be efficient for the production of correctly assembled and functional HepcH-FTH heteropolymer. Cysteine oxidation for the final refolded HepcH-FTH renatured in the proportion hepcidin/ferritin = 1:5, was carried out using the glutathione redox system (GSH/GSSG) as described by Jordan et al. [3].

#### **2.3 Cell culture**

Mouse monocyte–macrophage cell line J774 (Lombardy and Emilia Romagna Experimental Zootechnic Institute) was cultured as previously described by

Delaby et al. [4]. Briefly, cells were grown in DMEM (PAA Laboratories GmbH), 10% endotoxin-free fetal bovine serum (Euroclone), 0.04 mg/mL gentamicin (Euroclone), 2 mM l-glutamine (PAA Laboratories GmbH), and maintained at 37°C in 5% CO2. They (200,000 cells/well) were seeded onto 12- well plates, and after 24 h were grown for 12 h in presence of 100 μM ferric ammonium citrate (FAC) to induce ferroportin expression. The day after, cells were incubated with HepcH-FTH at final concentrations of 0.5 μM and 0.2 μM. Controls were cells without ferric ammonium citrate (FAC) treatment and cells incubated with native FTH homopolymer. Experiments were performed at 37°C for 30 min and 2 h. After this time, the supernatant was discarded and the cells washed with cold PBS and lysed using cold buffer (200 mM Tris–HCl at pH 8, 100 mM NaCl, 1 mM EDTA, 0.5% NP-40, 10% glycerol, 1 mM sodium fluoride, 1 mM sodium orthovanadate, complete protease inhibitor cocktail; Roche). Protein content was determined by colorimetric BCA assay (bicinchoninic acid assay, Pierce), 30 μg of total proteins were separated by native and denatured polyacrylamide gel electrophoresis and Western blotting, for the detection of proteins bound and internalized by the J774 cells, as well as the mass spectroscopy analysis were performed as described previously [5].
