Murine il 6

Murine C2C12 myoblasts and human Huh7 hepatoma cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 20% or 10% fetal bovine serum, respectively, non-essential amino acids, 100 U/mL penicillin and 100 μg/mL streptomycin. The medium was changed every 2–3 days, and the cells were passaged before reaching 80% confluence. For differentiation into myotubes, C2C12 cells were cultured for 2 weeks in media containing 2% fetal bovine serum. For differentiation into a cardiomyocyte-like lineage, C2C12 cells were cultured for 1 week in media containing 2% fetal bovine serum and supplemented with 500 nM retinoic acid. Where indicated, the cells were treated with the following recombinant proteins: 5 ng/mL human/mouse BMP6 and/or BMP2 (R&D Systems, Minneapolis, MN, USA), 20 ng/mL murine IL-6 (Cell Signaling, Danvers, MA, USA), or 10 ng/mL human IL-6 (Sigma-Aldrich, St. Louis, MI, USA).

Deferoxamine (DFO), FeSO₄ and ferric ammonium citrate (FAC) were purchased from Sigma-Aldrich. Following recombinant proteins were used: murine IL-6 (#5216, Cell Signaling), human IL-6 (#I1395, Sigma-Aldrich), human BMP6 (#507-BP, R&D Systems), and human holo-transferrin (#T4132, Sigma). Salicylaldehyde isonicotinoyl hydrazone (SIH) was a gift of the late Dr. Prem Ponka. Fe-SIH was prepared by mixing SIH with ferric citrate in 2:1 ratio [20 (link)].

Primary hepatocytes were isolated from mouse livers by using a modified two-step collagenase perfusion method [11 (link)]. The cells were treated with 20 ng/ml murine IL-6 (Cell Signaling).