Fig 2: Protein levels of regulators related to iron transport (NRAMP2, Natural resistance associated macrophage protein 2) and iron storage (ferritin) are decreased while expression of proteins participating in non transferrin bound iron (NTBI) uptake (ZIP14, Zinc importer protein 14) and iron export (ferroportin) does not differ in tumor-initiating cells (TICs)Protein levels of NRAMP2 (A), ferritin (B), zinc transporter protein 14 (C) and ferroportin (D) in the MCF-7 cell line (CTRL) and MCF-7 derived spheres (SPH) are shown. The protein expression was quantified by the image J software from 2 to 5 independent samples, standard error is SEM, p-values lower than 0.05 are denoted with a star and were calculated and plotted in GraphPad prism, using the unpaired t-test.

Fig 3: Identification of two new AHMIO1 patients. (A,B) Pedigrees of the two reported families. The probands are indicated with an arrow. Black symbols denote affected individuals, half-filled black symbols denote unaffected carriers. Individuals studied at the molecular level are indicated with the symbol #. IUFD: intrauterine fetal death. (C,D) Sanger sequencing validating the 762 + 35A > G (C) and 223G > A (D) mutations. The sequence of the respective probands is displayed, as well as that of a control (Ctr) and a relative (1-I.2 and 2-I.1, respectively). The reference sequences used are based on NM_000617.3 for SLC11A2 and on NP_000608.1 for DMT1. Hom: homozygous. Het: heterozygous.

Fig 4: DMT1 G75 residue analysis. (A) Partial amino acid sequence alignment of DMT1 protein in 20 species in the vicinity of DMT1 Glycine75 (G75), indicated with an arrow. RefSeq accession numbers are reported for each sequence except for E. coleocola, for which a Uniprot reference is given. Below, the alignment a star (*) indicates 100% conservation of the amino acid, while colons and dots indicate amino acids with strong and weak similarity, respectively. (B) Structural model of the DMT1 G75R mutation. The reference (left) and the mutated (right) residues are indicated as Gly-75 and Arg-75, respectively. TM: transmembrane domain.

Fig 5: PIEZO1 mediates ionizing radiation-induced pulmonary endothelial cell ferroptosis by increasing intracellular calcium concentration and calpain activity. (A) Intracellular Ca2+ measured using Fluo4-AM (5 mM) across groups. Histograms showing differences in Ca2+ concentrations across groups. (B) Calpain activity assay in HULEC-5a cell pre-treated with Yoda1 (2.5 µM) or GsMTx4 (5 µM) for 24 h before ionizing radiation (IR). (C) Lipid peroxidation assessment in HULEC-5a cells pre-treated with PD151746 (20 µM) for 24 h before IR. (D) ROS measurement in HULEC-5a cell pre-treated with PD151746 (20 µM) for 24 h before IR or without radiation. (E) Lipid peroxidation assessment in HULEC-5a cells pre-treated with PD151746 (20 µM) and/or Yoda1 for 24 h. The Lipid peroxidation level of the control group is the same result shown in Figure 3C. The grouping experiment belongs to single batch of experiment. (F) ROS measurement in HULEC-5a cells pre-treated with PD151746 (20 µM) and/or Yoda1 for 24 h. (G) Western blotting analysis of ACSL4, SLC7A11, DMT1, and GPX4 expression in HULEC-5a cell pretreated with PD151746 (20 µM) for 24 h followed by IR and/or treatment with Yoda1. Data were plotted as means ± SEM. n = 3 independent repeats. **, p < 0.01 vs control. &&, p < 0.01 vs PD151746. ##, p < 0.01 vs GsMTx4. $$, p < 0.01 vs Yoda1; %%, p < 0.01 vs IR.