Holo ferritin

Animal experiments were approved by the Institutional Animal Care and Use Committee at Massachusetts General Hospital (MGH). To generate mice with deletion of Tfrc in endothelial cells, C57BL/6J mice harboring LoxP-flanked alleles of Tfrc (Jackson Laboratory 028363) were bred to C57BL/6N mice expressing Cre recombinase under the control of an endothelial cell-specific Stabilin-2 (Stab2) promoter^{21 (link)} (generously provided by Cyrill Géraud, Mannheim, Germany) to produce Tfrc^fl/fl;Stab2-Cre+ and Tfrc^fl/fl;Stab2-Cre−littermate controls. For Cre reporter studies, Stab2-Cre+ mice were bred to B6.Cg-Gt(ROSA)26Sortm9(CAG-tdTomato)Hze/J (Jackson Laboratory 007909) Cre reporter mice.
Mice were housed in a temperature and humidity-controlled barrier facility on a 12-hour light-dark schedule and fed ad libitum with standard rodent chow (Prolab RMH 3000; LabDiet, 380 ppm iron). To model limited iron availability, mice were fed purified iron-limited diet (Envigo-Teklad TD190546, 20 ppm iron from ferric citrate) from 3–12 weeks of age. Iron content of the purified diets was confirmed by ICP-MS (Veterinary Diagnostic Laboratory). For ferritin injections, juvenile 5-week-old mice received a single intraperitoneal injection of 50 μg/g holo-ferritin (Sigma F4503) or solvent (PBS) for 6 hours.

Freshly isolated primary LECs were purchased from Cell Biologics (CD1017) and cultured on gelatin in complete growth media containing 5% fetal calf serum (Cell Biologics M1168) at 37 °C in a 5% CO₂ 95% air atmosphere. BMP6 induction experiments were performed up to passage 4–8 to maintain iron-mediated BMP6 responsiveness.
For iron depletion experiments, cells were treated with complete media supplemented with 100 μM deferoxamine (Sigma D9533) for 24 hours. For iron loading experiments, cells were serum starved for 8–16 hours prior to treatment with 200 μg/mL ferric ammonium citrate (Sigma F5879) for 6–24 hours, 30 μM holo-transferrin (Sigma T0665) or apo-transferrin (Sigma T1147) for 24 hours, or 100 nM holo-ferritin (Sigma F4503) or apo-ferritin (Sigma 178440) for 24 hours.
For gene knockdown experiments, cells were transfected with 40 nM non-targeting control siRNA (Invitrogen 4390846) or siRNA targeting Tfrc (Invitrogen s75457) using Lipofectamine RNAiMAX (Invitrogen) per the manufacturer’s instructions for 48 hours prior to iron treatment as described above. Gene knockdown was confirmed by qRT-PCR and immunoblotting.