Fig 2: Application of a cell-based model to assess the metformin and iron chelation treatment on iron misregulation and glucose dyshomeostasis(A) Schematic of cell stimulations. Cells were grown for 48 h in either low glucose (1 g/L) or high glucose (10 g/L) high iron media (additional 10 μM). Cells were then treated with either metformin (2 mM) (green octagon) or deferoxamine (25 μM) (purple diamond), an iron chelator.(B–F) Representative western blot of cell stimulations for transferrin receptor (TFRC) (B), ferritin heavy chain (FTH1) (C), fatty acid synthase (FAS) (D), sterol regulatory binding protein 1 (SREBP-1) (E), and glucose transporter 2 (GLUT2) (F).(G–K) Densitometry analysis of western blot for FAS (G), precursor form of SREBP-1 (∼125 kDa) (H), GLUT2 (I), TFR1 (J), and FTH1 (K) in stimulated cells (n = 3). Loading controls in panels B, C, and D are from the same membranes as are loading controls in panels E and F as these proteins were detected via sequential probing of a single gel. Full uncropped blots with molecular weight markers for all western blot panels presented in this figure are provided in Figure S10. Data displayed as Mean ± SEM. α-tubulin was utilized as a loading control for all western blot analyses. One-tailed t test was used for all statistical analysis. ∗p-value <0.05, ∗∗p-value <0.01, ∗∗∗p-value <0.001, ∗∗∗∗p-value <0.0001.

Fig 3: (A) Ferrous iron (Fe2+) contents and transferrin receptor (TFRC) levels, (B) glutathione peroxidase 4 (GPX4) activities, reduced glutathione (GSH), and 4-hydroxynonenal (4-HNE) levels in lungs of mice at two time points after cecal ligation and puncture (CLP). Calcitriol treatment reduced Fe2+, TFRC, and 4-HNE levels, and enhanced GSH levels and GPX4 activities in ovariectomized mice with sepsis. OB, mice with a sham-ovariectomy operation and a high-fat diet (HFD) (n = 8); OVSS, mice with an ovariectomy, an HFD, and then cecal ligation and puncture (CLP) was performed, and saline was injected after CLP, sacrificed on either 24 h (OVSS-24) or 72 h (OVSS-72) (n = 8 at each time point); and OVSD, mice with an ovariectomy, an HFD, and CLP, and calcitriol was injected after CLP, sacrificed on either 24 h (OVSD-24) or 72 h (OVSD-72) (n = 8 at each time point). Values are expressed as the mean ± SEM. Comparisons among experimental groups were analyzed by a two-way ANOVA followed by the Bonferroni post hoc test. +Significantly differs from the OVSS group on 72 h. ⁣∗Significantly differs from the OVSS group at the same time point (p < 0.05).

Fig 4: HGD induces hepatic iron overload(A) Inductively coupled plasma mass spectrometry (ICP-MS) analysis of liver tissue of HGD and vehicle mice. Samples were normalized by tissue weight. (n = 6) (B) Real-time quantitative PCR of iron regulators in the liver of HGD and vehicle mice. Gene expression analysis was performed for divalent metal transporter 1 (DMT1), ferroportin (FPN1), transferrin receptor 1 (TFRC), ferritin heavy chain (FHC), ferritin light chain (FLC), hepcidin (HAMP), iron-responsive element-binding protein 1 (IRP1), and iron-responsive element-binding protein 2 (IRP2). (n = 6) (C) Representative western blot of transferrin receptor (TFRC) in the liver of HGD and vehicle mice.(D) Representative western blot of ferritin heavy chain 1 (FTH1) in the liver of HGD and vehicle mice.(E)Representative western blot of divalent metal transporter 1 (DMT1) in the liver of HGD and vehicle mice.(F) Representative western blot of ferroportin (FPN1) in the liver of HGD and vehicle mice.(G) Densitometry analysis of TFRC western blot for HGD and vehicle mice (n = 6).(H) Densitometry analysis of FTH1 western blot for HGD and vehicle mice (n = 6).(I) Densitometry analysis of DMT1 western blot for HGD and vehicle mice (n = 6).(J) Densitometry analysis of FPN1 western blot for HGD and vehicle mice (n = 6). Full uncropped blots with molecular weight markers for all western blot panels presented in this figure are provided in Figure S9. Data displayed as Mean ± SEM. α-tubulin was utilized as a loading control for all western blot analysis. One-tailed t test was used for all statistical analyses. ∗p-value <0.05, ∗∗p-value <0.01, ∗∗∗p-value <0.001, ∗∗∗∗p-value <0.0001.

Fig 5: TfR1 interacts with iRhom2 at the cell surface, with the cytosolic N terminus of iRhom2 serving as a determinant of this binding.a, Schematic representation of the iRhom2 variants used in this study. Shown are different iRhom2 mutations that trap iRhom2 at differing stages in the secretory pathway. The murine iRhom2_W538S does not exit the ER, while murine iRhom2_KDEL (added C-terminal signal sequence KDEL) is sorted back to the ER25. By contrast, murine iRhom2_NOSL (S644N, P691A and A692S) and murine iRhom2_GRIP (added C-terminal GRIP domain) do not leave the Golgi apparatus. All other variants used in this study successfully reach the cell surface (wt murine iRhom1, wt murine iRhom2, murine iRhom2_pDEAD46, murine iRhom2_nd197, murine iRhom2_nd349, murine iRhom2_nd370, murine iRhom2_cub, wt human iRhom1, wt human iRhom2, human iRhom2_ndTOC and human iRhom2_I186T41). In the murine iRhom2_pDEAD variant, all potential phosphorylation sites in the iRhom N terminus were mutated to alanine. The murine iRhom2_nd197 variant lacks amino acids 1–197, while the murine iRhom2_cub variant lacks amino acids 1–268. The two other deletions extend beyond the region deleted in the cub variant, with murine iRhom2_nd349 lacking amino acids 1–349 and murine iRhom2_nd370 lacking nearly the entire N terminus (amino acids 1–370). The N-terminal deletion variant of the human iRhom2 spanning the TOC mutation sides (human iRhom2_ndTOC) only removes a small region of the N terminus (Δ182–196). b–n, The HEK293 cells used in this study were either transduced with the indicated iRhom variants or GFP (ctr) as a negative control. If not stated otherwise, wt HEK293 cells were used. In (b), additionally, ADAM10/ADAM17 dKO cells (ADAM10/ADAM17 dKO) were used (n = 4), and in (m), iRhom1/iRhom2 dKO cells (iRhom1/iRhom2 dKO) were used (n = 3). CoIPs were performed to analyze the binding between TfR1 and iRhom variants. The iRhom variants with an HA tag were used as bait, and the TfR1 binding was quantified by calculating the levels of coprecipitated TfR1 (prey) relative to the precipitated iRhom variants (bait) (ratio bound TfR1/iRhom). To analyze TfR1–iRhom binding at the cell surface, a surface coIP was performed (Methods) (n = 5) (f). The samples were prepared with reducing (+β-mercaptoethanol (β-meEtOH)) or nonreducing (−β-meEtOH) conditions to compare the TfR1 monomer or TfR1 dimer binding to wt murine iRhom2 (n = 3) (h). The corresponding lysate controls of all coIPs are shown in Supplementary Fig. 2. The bands marked with an asterisk indicate a fragment of the antibody used for immunoprecipitation. The higher iRhom band represents the full-length iRhom, and the lower iRhom band represents a cleaved fragment of iRhom40. n = 4 (c), n ≥ 3 (d and e), n = 5 (g), n = 3 (i and j), n = 4 (k and l), n = 3 (n). Data are presented as mean + s.d. from at least three independent experiments. Significant differences were indicated by asterisks (*P < 0.05, **P < 0.01, ***P < 0.001). b, Representative western blot of coIP of wt HEK239 and ADAM10/ADAM17 KO cells with control (GFP), wt murine iRhom2, murine iRhom2_NOSL. c, Quantification of (b). d, Representative western blot of coIP of wt HEK239 cells with control (GFP), wt murine iRhom2, murine iRhom2_KDEL, murine iRhom2_W538S, murine iRhom2_GRIP. e, Quantification of (d). f, representative western blot of surface coIP of wt HEK239 cells with control (GFP), wt murine iRhom1, murine iRhom2. g, Quantification of (f). h, Representative western blot of coIP of wt HEK239 cells with control (GFP), wt murine iRhom2. i, Representative western blot of coIP of wt HEK239 cells with control (GFP), wt murine iRhom2, murine iRhom2_nd197, murine iRhom2_nd349, murine iRhom2_nd370, murine iRhom2_cub. j, Quantification of (i). k, Representative western blot of coIP of wt HEK239 cells with control (GFP), wt murine iRhom2, murine iRhom2_ndTOC, murine iRhom2_I186T. l, Quantification of (k). m, Representative western blot of coIP of iRhom1/iRhom2 dKO HEK293 cells with control (GFP), wt murine iRhom2, murine iRhom2_pDEAD. n, Quantification of (m).