Ferric citrate

DMEM (high glucose, [4.5 g/L]), NaCl, FBS, and a BCA protein assay kit (Pierce, Rockford, IL, USA) were purchased from Euroclone (Milan, Italy); Na₃PO₄, MgSO₄, NaH₂PO₄, KH₂PO₄, and KCl were purchased from Carlo Erba (Milan, Italy); ferric citrate, Trypsin-EDTA-Solution (T4174) and Collagenasi type IA (C9891) were purchased from Sigma (St. Louis, MO, USA); the primary antibody for Axl (sc-1097) and GAS6 (N-20 sc-1936) were purchased from Santa Cruz (Heidelberg, Germany); the primary antibody for LC3-II was purchased from Cell Signalling (Danvers, MA, USA), and the anti-rabbit secondary antibody was purchased from GeneTex (Irvine, CA, USA); the anti-goat secondary antibody (ab6741) was purchased from Abcam (Cambrige, UK); Hepes Buffer Solution, PVDF membrane Invitrogen/Applied Biosystem (Milan, Italy); PE Annexin V Apoptosis Detection Kit I (559763) was purchased from Bioscience. An ApopTag Red In Situ Apoptosis Detection kit S7165 was purchased from Chemicon. All other reagents were obtained from Sigma (St. Louis, MO, USA).

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.

MOLM-14, OCI-AML2, HL-60, SET2, MV4-11, K562, THP-1, UT7-EPO, SKM1, NB4 and KASUMI-1 AML cell lines were used. Patients provided written informed consent in accordance with the Declaration of Helsinki. Bone marrow (BM) samples were obtained from five patients with newly diagnosed AML (characteristics provided in the Supplementary Table S1). Cells were cultured in RPMI with glutamine (Gibco61870, Life Technologies® Saint Aubin, France) supplemented with 10% fetal bovine serum (FCS) and 4 mM glutamine. Ferric Citrate (FAC) was purchased from Sigma. DHA, Ferrostatin-1, Deferoxamin (DFO), Deferasirox (DFX), QVD-OPH, APR-246, VPS34-in1, erastin, FIN56, and RSL3 for the in vitro study were sourced from Selleckchem (Houston, TX). L -C-Propargylglycine (PPG), Chloroquine and doxycycline were obtained from Sigma–Aldrich (Saint-Louis, MO). FINO2 was purchased from Cayman Chemicals (Ann Arbor, MI).

Erastin (E7781, Sigma–Aldrich, Saint-Quentin-Fallavier, France) mother solution was prepared at an initial concentration of 10 mM in 100% DMSO (FLUKA), aliquoted and stored at −80 °C. Ferric citrate (F3388-Sigma–Aldrich, Saint-Quentin-Fallavier, France) mother solution was prepared at an initial concentration of 25 mM in warm PBS (37 °C) and stored at 4 °C. Deferoxamine (D0160000, European Pharmacopia, Starsbourg, France) mother solution was prepared at an initial concentration of 30 mM in PBS, aliquoted, and stored at −80 °C. Deferiprone (Y0001976, European Pharmacopia, Starsbourg, France) mother solution was prepared at an initial concentration of 134 mM in warm ultra-pure water, aliquoted, and stored at −80 °C.

Thirty (n = 6/group) male Sprague-Dawley (SD) rats aged 8 weeks were randomly assigned to five groups: (1) a control group fed a normal diet with 36.7 mg ferric iron/kg diet (14.9% protein, 9.4% fat, 75.7% carbohydrates; total energy: 3.81 kcal/g diet), (2) an HFD group with 36.7 mg ferric iron/kg diet (14.8% protein, 50.0% fat, 38.7% carbohydrates; total energy: 4.80 kcal/g diet), (3) an HFD group plus 0.25 g ferric iron/kg diet (14.8% protein, 50.0% fat, 38.7% carbohydrates; total energy: 4.80 kcal/g diet), (4) an HFD group plus 1 g ferric iron/kg diet (14.8% protein, 50.0% fat, 38.7% carbohydrates; total energy: 4.80 kcal/g diet), and (5) an HFD group plus 2 g ferric iron/kg diet (14.8% protein, 50.0% fat, 38.7% carbohydrates; total energy: 4.80 kcal/g diet) for 12 weeks. All animal procedures were conducted under a protocol approved by the Animal Ethical Committee of Taipei Medical University (LAC-2014-0254). Ferric citrate was purchased from Sigma-Aldrich (St. Louis, MO, USA). The AIN-93 M standard diet was purchased from MP Biomedicals (Fisher Scientific; New Taipei City, Taiwan).

Weanling male Sprague-Dawley rats (Charles River Laboratories) were randomized (n = 6/group) to receive either iron-deficient (FeD), iron-adequate (FeA), or iron-overloaded (FeO) diets. Purified diets were prepared according to the AIN-93G formulation, but with no added iron (FeD), 35 mg/kg ferric citrate (FeA), or 2% carbonyl iron (Sigma-Aldrich) (FeO). Iron contents of the diets, as determined by inductively coupled plasma mass spectroscopy (ICP-MS), were 5 ppm (FeD), 36 ppm (FeA), and 20,275 ppm (FeO). Diets were also modified to contain Avicel® microcrystalline cellulose instead of cellulose (to minimize contaminant iron) and 20% sucrose instead of 10% sucrose (while reducing the amount of cornstarch accordingly) [11] (link). The amount of sucrose was increased in order to make the iron-loaded diet more palatable. After 3 weeks of feeding, overnight-fasted rats were sacrificed by exsanguination from the descending aorta. Blood was collected into heparinized syringes and then centrifuged to obtain plasma. Pancreases were quickly harvested, immediately frozen in liquid nitrogen, and maintained at −80°C for subsequent analyses. Animal experiments were approved by the Institutional Animal Care and Use Committee at the University of Florida (Protocol # 201101613).