Fig 2: Generation of KO and re-expressed cell lines (related to Fig. 4).a, b. Representative IB analysis of generated re-expressing cell lines of HMOX2 WT, HW/GW mutant, or K68R mutant. Re-expressing cell lines of HMOX2 WT, K68R mutant, or HW/GW mutant was generated by stably expressing each HMOX2 in HMOX2 KO #1 cells. α-tubulin serves as a loading control. c. Stably expressed HMOX2 localize to the ER. Re-expressing cell lines of HMOX2 WT, HW/GW mutant, or K68R mutant were analyzed by immunofluorescence with anti-HA, anti-Calnexin, and anti-Tom20 antibodies to visualize the localization of HMOX2, ER, and mitochondria, respectively. Three-slice images were reconstructed, and the right panels show enlarged images of the boxed region. Scale bar, 10 µm. d, e. Representative IB analysis of generated PDZD8 KO and VAPB KO HeLa cells. Each KO cell lines were generated by the CRISPR–Cas9 system with two different gRNAs shown in Supplementary table. The cell lines marked with red squares were used for the PDZD8 KO cell experiments, shown in Fig. 4g. α-tubulin and Vinculin serve as loading controls. Data are representative of three independent experiments with similar results. Unprocessed blots are available in source data. Source data

Fig 3: Haem-derived iron is essential for mitochondrial respiration.a, Schematic of the experimental design to evaluate the role of haem-derived iron in mitochondria. Ctrl, control; HD, haem depletion; SA, succinylacetone; FAC, ferric ammonium citrate. b,c, Rieske and SDHB protein levels are stabilized in a haem-dependent manner. b, IB analysis of lysates from cells cultured under the four conditions shown in a. α-tubulin serves as loading control. c, Rieske and SDHB protein levels, quantified and normalized by α-tubulin from b. Fold changes relative to Ctrl cells are shown. Data are presented as mean ± s.d. from three independent experiments. One-way ANOVA with Tukey’s multiple comparisons; NS, not significant; ****P < 0.0001, **P < 0.01, *P < 0.05. d, Mitochondrial respiration is maintained in a haem-dependent manner. Basal respiration was measured using an Oxygraph-2k instrument in cells cultured under the four conditions shown in a. Data are presented as mean ± s.d. from three independent experiments. One-way ANOVA with Tukey’s multiple comparisons; NS, not significant; **P < 0.01, *P < 0.05. e, HMOX1-mediated haem degradation does not affect Rieske and SDHB protein stability upon haemin treatment. Left: IB analysis of lysates from cells cultured under the indicated conditions. SDHA, ATP5A and Tom20 serve as mitochondrial protein controls. α-tubulin and vinculin serve as the loading control. Right: Rieske and SDHB protein levels were quantified and normalized by vinculin from the left panel. Fold changes relative to Ctrl cells are shown. Data are presented as mean ± s.d. from three independent experiments. One-way ANOVA with Tukey’s multiple comparisons; NS, not significant; **P < 0.01, *P < 0.05. f, HMOX2-mediated haem degradation stabilizes Rieske and SDHB protein levels upon haemin treatment. Top: IB analysis of lysates from cells cultured under the indicated conditions. α-tubulin and vinculin serve as loading control. Bottom: Rieske and SDHB protein levels were quantified and normalized by vinculin from the top panel. Fold changes relative to Ctrl cells are shown. Data are presented as mean ± s.d. from three independent experiments. One-way ANOVA with Tukey’s multiple comparisons; NS, not significant; ***P < 0.001, *P < 0.05. Source numerical data and unprocessed blots are available as Source data. Illustrations in a created in BioRender; Oshio, H. https://BioRender.com/v093d2u (2026).Source data

Fig 4: Subcellular localization and MITOL-mediated ubiquitination of HMOX2 mutants (related to Fig. 3).a. HMOX2 mutants localize to the ER. HeLa cells transfected with the indicated vectors were analyzed by immunofluorescence with anti-HA, anti-Calnexin, and anti-Tom20 antibodies to visualize the localization of HMOX2, ER, and mitochondria, respectively. Three-slice images were reconstructed, and the right panels show enlarged images of the boxed region. Scale bar, 10 μm. b. MITOL-mediated ubiquitination of HMOX2 mutants. WT HeLa cells transfected with the indicated vectors were analyzed by IP-IB assay to compare ubiquitination levels of WT and mutants of HA-HMOX2. Conditions labeled “(-)” indicate transfection with an empty vector. WT, wild-type HMOX2; H45W/G159W, HMOX2 H45W/G159W mutant (lacking haem-degrading activity and haem in the catalytic core); G163H, HMOX2 G163H mutant (lacking haem-degrading activity, high haem affinity in the catalytic core); C265A, HMOX2 C265A mutant (lacking haem transfer activity). Data are representative of three independent experiments with similar results. Unprocessed blots are available in source data. Source data

Fig 5: Conditions enhancing MITOL-mediated HMOX2 ubiquitination (related to Fig. 4).a. IB analysis of HeLa cells treated for 24 h with 1 mM succinyl acetone (SA), 1 mM 5-aminolevulinic acid (5-ALA), 20 µM hemin, 50 µM deferoxamine (DFO), or 1 mM ferric ammonium citrate (FAC). α-tubulin serves as a loading control. b. DFO treatment enhances MITOL-mediated HMOX2 ubiquitination. HeLa cells transfected with the indicated vectors were treated with the compounds listed in (a). IP-IB analysis compared ubiquitination levels in DMSO-treated vs. stimulated conditions. FAC was used at 10 µM to avoid inhibition of transfection. Conditions labeled “(-)” indicate transfection with an empty vector. FTH1 is used as a marker of iron accumulation. α-tubulin serves as a loading control. c. DFO treatment enhances MITOL-mediated HMOX2 ubiquitination. 50 µM DFO was treated with transfection with the indicated vectors 20 h before the IP-IB assay. IP-IB analysis compared ubiquitination levels in DFO treated cells with DMSO-treated cells. Conditions labeled “(-)” indicate transfection with an empty vector. α-tubulin serves as a loading control. d. Knockdown of MFRN1 enhances HMOX2 ubiquitination by MITOL. HeLa cells were transfected with scrambled siRNA (sc) or siMFRN1 for 48 h, followed by transfection with the indicated vectors 24 h before IP-IB analysis. Conditions labeled “(-)” indicate transfection with an empty vector. Tom20 serves as a mitochondrial protein control. α-tubulin and Vinculin serve as loading controls. e. DFO treatment enhances MITOL-mediated ubiquitination of endogenous HMOX2. 3×HA–HMOX2 knock-in cells were transfected with siMITOL for 48 h, followed by treatment with 50 µM DFO for 24 h prior to IP–IB analysis. α-tubulin serves as a loading control. Data are representative of three independent experiments with similar results. Unprocessed blots are available in source data. Source data