Autophagy-deficient BMDMs can form ferric ferritin and have high ferroportin levels. (A) Protein expression (i) and ferric ferritin (ii) in nontreated or treated BMDMs with FAC, holo-TF, or oRBCs. BMDMs plated (10⁵) onto 96-well plates were treated for 16 hours either with FAC (100 μM), holo-TF (30 μM), or oRBCs (oRBCs to cells, 5:1). (Ai) Immunoblotting of Atg5 (Atg5/Atg12 complex is shown), p62, Fpn, Ft-H, HO-1, and β-actin as the loading controls. (Aii) Staining of ferric iron loaded in ferritin on native polyacrylamide gel electrophoresis (PAGE) gels in BMDMs after the indicated treatments; 25 ng of native proteins from cytosolic lysates was loaded in each lane of a nondenaturing PAGE gel, and after electrophoresis, the gel was directly stained with Perl’s staining solution and enhanced with DAB (representative images from n = 3 experiments are shown; quantitative analysis of the western blots is given in supplemental Table 3). (B) Calcein quenching assay for labile iron pool (LIP) measurement, ROS levels, and membrane TfR1 expression. Cells were plated (10⁵) in 96-well plates and treated for 16 hours with FAC (100 μM), holo-TF (30 μM), or oRBCs (oRBCs to cells, 5:1). For intracellular LIP measurement based on calcein quenching and the ROS assay, after addition of Calcein-AM (calcein quenching assay) or H₂DCFDA (ROS assay), fluorescence intensity was measured (representative analysis of n = 3 independent experiments is shown). For calcein, the mean fluorescence intensity of treated cells was normalized to that of untreated control cells. ROS production is represented as the percentage relative to that in untreated control cells. For evaluation of TfR1 membrane expression, cells were incubated with BV510 anti-CD45, phycoerythrin-Cy7 F4/80, and eFluor450 CD71 anti-mouse antibodies and analyzed by flow cytometry.