Plp 4392 s

Mouse PASMCs were treated with mouse HAMP peptide (PLP-4434-s, Peptides International) while human PASMCs were treated with human HAMP peptide (PLP-4392-s, Peptides International) at a final concentration of 0.5 μmol/L for 12–16 h. Ferric citrate (F3388, Sigma-Aldrich) was dissolved in cell growth medium at a final concentration of 200 μmol/L. Deferroxamine (D9533, Sigma-Aldrich) was used at 100 μmol/L. Human and mouse hamp siRNA (Silencer Select assay code S195324 and S96551, respectively, Thermo Fisher Scientific) were transfected into PASMCs at a concentration of 50 nmol/L using Lipofectamine 2000 (11668027, Thermo Fisher Scientific) according to manufacturer’s instructions.

Human hepcidin (PLP-4392-S; Peptide institute, Osaka, Japan), 1,25(OH)₂D₃ (Cayman Chemical, Ann Arbor, MI, USA), L-Ascorbic acid 2-phosphate sesquimagnesium salt hydrate (L-Ascorbic; A8960; Sigma-Aldrich, Saint Louis, MO, USA) and calcium chloride (CaCl₂; Sigma-Aldrich) were used. Ferric ammonium citrate (FAC; Sigma-Aldrich) was utilized as a donor of ferric iron.