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  • Expert Recommendation
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Recommendations for robust and reproducible research on ferroptosis

Nature Reviews Molecular Cell Biology volume 26, pages 615–630 (2025)Cite this article

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Abstract

Ferroptosis is a necrotic, non-apoptotic cell death modality triggered by unrestrained iron-dependent lipid peroxidation. By unveiling the regulatory mechanisms of ferroptosis and its relevance to various diseases, research over the past decade has positioned ferroptosis as a promising therapeutic target. The rapid growth of this research field presents challenges, associated with potentially inadequate experimental approaches that may lead to misinterpretations in the assessment of ferroptosis. Typical examples include assessing whether an observed phenotype is indeed linked to ferroptosis, and selecting appropriate animal models and small-molecule modulators of ferroptotic cell death. This Expert Recommendation outlines state-of-the-art methods and tools to reliably study ferroptosis and increase the reproducibility and robustness of experimental results. We present highly validated compounds and animal models, and discuss their advantages and limitations. Furthermore, we provide an overview of the regulatory mechanisms and the best-studied players in ferroptosis regulation, such as GPX4, FSP1, SLC7A11 and ACSL4, discussing frequent pitfalls in experimental design and relevant guidance. These recommendations are intended for researchers at all levels, including those entering the expanding and exciting field of ferroptosis research.

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Fig. 1: Overview of intracellular ferroptosis regulatory pathways.
Fig. 2: Experimental procedures for determining ferroptosis.
Fig. 3: Experimental factors affecting the sensitivity of cells to undergo ferroptosis.

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References

  1. Dixon, S. J. et al. Ferroptosis: an iron-dependent form of nonapoptotic cell death. Cell 149, 1060–1072 (2012). This seminal study coined the term ferroptosis, galvanizing various lines of research at the intersection of redox biology, iron and lipid homeostasis and cell death.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Dixon, S. J. & Olzmann, J. A. The cell biology of ferroptosis. Nat. Rev. Mol. Cell Biol. 25, 424–442 (2024).

    Article  PubMed  PubMed Central  Google Scholar 

  3. Jiang, X., Stockwell, B. R. & Conrad, M. Ferroptosis: mechanisms, biology and role in disease. Nat. Rev. Mol. Cell Biol. 22, 266–282 (2021).

    Article  PubMed  PubMed Central  Google Scholar 

  4. Ingold, I. et al. Selenium utilization by GPX4 is required to prevent hydroperoxide-induced ferroptosis. Cell 172, 409–422.e421 (2018).

    Article  CAS  PubMed  Google Scholar 

  5. Badgley, M. A. et al. Cysteine depletion induces pancreatic tumor ferroptosis in mice. Science 368, 85–89 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Zhao, J. et al. Human hematopoietic stem cell vulnerability to ferroptosis. Cell 186, 732–747.e716 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Friedmann Angeli, J. P. et al. Inactivation of the ferroptosis regulator Gpx4 triggers acute renal failure in mice. Nat. Cell Biol. 16, 1180–1191 (2014). This study bdemonstrates that genetic deletion of Gpx4 induces ferroptosis, leading to acute renal failure in mice, and identifies Lip-1 as a potent ferroptosis inhibitor.

    Article  CAS  PubMed  Google Scholar 

  8. Viswanathan, V. S. et al. Dependency of a therapy-resistant state of cancer cells on a lipid peroxidase pathway. Nature 547, 453–457 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Hangauer, M. J. et al. Drug-tolerant persister cancer cells are vulnerable to GPX4 inhibition. Nature 551, 247–250 (2017). This seminal work demonstrates that drug-tolerant persister cancer cells depend on GPX4 for survival and that inhibiting GPX4 induces ferroptotic cell death in these cells, thereby preventing tumour relapse in vivo.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Yang, W. S. et al. Regulation of ferroptotic cancer cell death by GPX4. Cell 156, 317–331 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Wang, W. et al. CD8+ T cells regulate tumour ferroptosis during cancer immunotherapy. Nature 569, 270–274 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Wu, J. et al. Intercellular interaction dictates cancer cell ferroptosis via NF2-YAP signalling. Nature 572, 402–406 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Ubellacker, J. M. et al. Lymph protects metastasizing melanoma cells from ferroptosis. Nature 585, 113–118 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Kim, R. et al. Ferroptosis of tumour neutrophils causes immune suppression in cancer. Nature 612, 338–346 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Kalkavan, H. et al. Sublethal cytochrome c release generates drug-tolerant persister cells. Cell 185, 3356–3374.e3322 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Hambright, W. S., Fonseca, R. S., Chen, L., Na, R. & Ran, Q. Ablation of ferroptosis regulator glutathione peroxidase 4 in forebrain neurons promotes cognitive impairment and neurodegeneration. Redox Biol. 12, 8–17 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Linkermann, A. et al. Synchronized renal tubular cell death involves ferroptosis. Proc. Natl Acad. Sci. USA 111, 16836–16841 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Berndt, C. et al. Ferroptosis in health and disease. Redox Biol. 75, 103211 (2024).

    Article  PubMed  PubMed Central  Google Scholar 

  19. Nakamura, T. & Conrad, M. Exploiting ferroptosis vulnerabilities in cancer. Nat. Cell Biol. 26, 1407–1419 (2024).

    Article  PubMed  Google Scholar 

  20. Schwab, A. et al. Zeb1 mediates EMT/plasticity-associated ferroptosis sensitivity in cancer cells by regulating lipogenic enzyme expression and phospholipid composition. Nat. Cell Biol. 26, 1470–1481 (2024).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Hirata, Y. et al. Lipid peroxidation increases membrane tension, Piezo1 gating, and cation permeability to execute ferroptosis. Curr. Biol. 33, 1282–1294.e1285 (2023).

    Article  CAS  PubMed  Google Scholar 

  22. Wiernicki, B. et al. Excessive phospholipid peroxidation distinguishes ferroptosis from other cell death modes including pyroptosis. Cell Death Dis. 11, 922 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Seiler, A. et al. Glutathione peroxidase 4 senses and translates oxidative stress into 12/15-lipoxygenase dependent- and AIF-mediated cell death. Cell Metab. 8, 237–248 (2008). The authors describe a lethal phenotype after deletion of Gpx4, iconically describing the observed cell death as ‘a-yet-unrecognized cell death’.

    Article  CAS  PubMed  Google Scholar 

  24. Thomas, J. P., Maiorino, M., Ursini, F. & Girotti, A. W. Protective action of phospholipid hydroperoxide glutathione peroxidase against membrane-damaging lipid peroxidation. In situ reduction of phospholipid and cholesterol hydroperoxides. J. Biol. Chem. 265, 454–461 (1990).

    Article  CAS  PubMed  Google Scholar 

  25. Sato, H., Tamba, M., Ishii, T. & Bannai, S. Cloning and expression of a plasma membrane cystine/glutamate exchange transporter composed of two distinct proteins. J. Biol. Chem. 274, 11455–11458 (1999). In this milestone work, the authors clone and characterize the plasma membrane cystine/glutamate exchange transporter, system xc−.

    Article  CAS  PubMed  Google Scholar 

  26. Doll, S. et al. FSP1 is a glutathione-independent ferroptosis suppressor. Nature 575, 693–698 (2019). This work identifies FSP1 as a novel, glutathione-independent suppressor of ferroptosis, demonstrating that FSP1 reduces extramitochondrial coenzyme Q10 to prevent lipid peroxidation and cell death.

    Article  CAS  PubMed  Google Scholar 

  27. Bersuker, K. et al. The CoQ oxidoreductase FSP1 acts parallel to GPX4 to inhibit ferroptosis. Nature 575, 688–692 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Mishima, E. et al. A non-canonical vitamin K cycle is a potent ferroptosis suppressor. Nature 608, 778–783 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Kraft, V. A. N. et al. GTP cyclohydrolase 1/tetrahydrobiopterin counteract ferroptosis through lipid remodeling. ACS Cent. Sci. 6, 41–53 (2020).

    Article  CAS  PubMed  Google Scholar 

  30. Soula, M. et al. Metabolic determinants of cancer cell sensitivity to canonical ferroptosis inducers. Nat. Chem. Biol. 16, 1351–1360 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Ingold, K. U. & Pratt, D. A. Advances in radical-trapping antioxidant chemistry in the 21st century: a kinetics and mechanisms perspective. Chem. Rev. 114, 9022–9046 (2014).

    Article  CAS  PubMed  Google Scholar 

  32. Li, Y. et al. 7-Dehydrocholesterol dictates ferroptosis sensitivity. Nature 626, 411–418 (2024).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Freitas, F. P. et al. 7-Dehydrocholesterol is an endogenous suppressor of ferroptosis. Nature 626, 401–410 (2024).

    Article  CAS  PubMed  Google Scholar 

  34. Garcia-Bermudez, J. et al. Squalene accumulation in cholesterol auxotrophic lymphomas prevents oxidative cell death. Nature 567, 118–122 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Warner, G. J. et al. Inhibition of selenoprotein synthesis by selenocysteine tRNA[Ser]Sec lacking isopentenyladenosine. J. Biol. Chem. 275, 28110–28119 (2000).

    Article  CAS  PubMed  Google Scholar 

  36. Aldrovandi, M., Fedorova, M. & Conrad, M. Juggling with lipids, a game of Russian roulette. Trends Endocrinol. Metab. 32, 463–473 (2021).

    Article  CAS  PubMed  Google Scholar 

  37. Li, Z., Lange, M., Dixon, S. J. & Olzmann, J. A. Lipid quality control and ferroptosis: from concept to mechanism. Annu. Rev. Biochem. 93, 499–528 (2024).

    Article  PubMed  Google Scholar 

  38. Hirata, Y. & Mishima, E. Membrane dynamics and cation handling in ferroptosis. Physiology 39, 73–87 (2024).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Doll, S. et al. ACSL4 dictates ferroptosis sensitivity by shaping cellular lipid composition. Nat. Chem. Biol. 13, 91–98 (2017). Using a genome-wide CRISPR-based genetic screen, this study identified ACSL4 as a key determinant of ferroptosis sensitivity.

    Article  CAS  PubMed  Google Scholar 

  40. Kagan, V. E. et al. Oxidized arachidonic and adrenic PEs navigate cells to ferroptosis. Nat. Chem. Biol. 13, 81–90 (2017).

    Article  CAS  PubMed  Google Scholar 

  41. Xu, L., Davis, T. A. & Porter, N. A. Rate constants for peroxidation of polyunsaturated fatty acids and sterols in solution and in liposomes. J. Am. Chem. Soc. 131, 13037–13044 (2009).

    Article  CAS  PubMed  Google Scholar 

  42. Morgan, P. K. et al. A lipid atlas of human and mouse immune cells provides insights into ferroptosis susceptibility. Nat. Cell Biol. 26, 645–659 (2024).

    Article  CAS  PubMed  Google Scholar 

  43. Qiu, B. et al. Phospholipids with two polyunsaturated fatty acyl tails promote ferroptosis. Cell 187, 1177–1190.e1118 (2024).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Magtanong, L. et al. Exogenous monounsaturated fatty acids promote a ferroptosis-resistant cell state. Cell Chem. Biol. 26, 420–432.e429 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Zhang, H. L. et al. PKCβII phosphorylates ACSL4 to amplify lipid peroxidation to induce ferroptosis. Nat. Cell Biol. 24, 88–98 (2022).

    Article  CAS  PubMed  Google Scholar 

  46. Beatty, A. et al. Ferroptotic cell death triggered by conjugated linolenic acids is mediated by ACSL1. Nat. Commun. 12, 2244 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Tesfay, L. et al. Stearoyl-CoA desaturase 1 protects ovarian cancer cells from ferroptotic cell death. Cancer Res. 79, 5355–5366 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Rodencal, J. et al. Sensitization of cancer cells to ferroptosis coincident with cell cycle arrest. Cell Chem. Biol. 31, 234–248.e213 (2024).

    Article  CAS  PubMed  Google Scholar 

  49. Liang, D. et al. Ferroptosis surveillance independent of GPX4 and differentially regulated by sex hormones. Cell 186, 2748–2764.e2722 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Conrad, M. & Pratt, D. A. The chemical basis of ferroptosis. Nat. Chem. Biol. 15, 1137–1147 (2019).

    Article  CAS  PubMed  Google Scholar 

  51. Galy, B., Conrad, M. & Muckenthaler, M. Mechanisms controlling cellular and systemic iron homeostasis. Nat. Rev. Mol. Cell Biol. 25, 133–155 (2024).

    Article  PubMed  Google Scholar 

  52. Aron, A. T., Loehr, M. O., Bogena, J. & Chang, C. J. An endoperoxide reactivity-based FRET probe for ratiometric fluorescence imaging of labile iron pools in living cells. J. Am. Chem. Soc. 138, 14338–14346 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Hirayama, T., Miki, A. & Nagasawa, H. Organelle-specific analysis of labile Fe(II) during ferroptosis by using a cocktail of various colour organelle-targeted fluorescent probes. Metallomics 11, 111–117 (2019).

    Article  CAS  PubMed  Google Scholar 

  54. Dixon, S. J. & Lee, M. J. Quick tips for interpreting cell death experiments. Nat. Cell Biol. 25, 1720–1723 (2023).

    Article  CAS  PubMed  Google Scholar 

  55. Toyokuni, S. et al. Iron as spirit of life to share under monopoly. J. Clin. Biochem. Nutr. 71, 78–88 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Cheng, Z. et al. Ferroptosis resistance determines high susceptibility of murine A/J strain to iron-induced renal carcinogenesis. Cancer Sci. 113, 65–78 (2022).

    Article  CAS  PubMed  Google Scholar 

  57. Zheng, H., Jiang, L., Tsuduki, T., Conrad, M. & Toyokuni, S. Embryonal erythropoiesis and aging exploit ferroptosis. Redox Biol. 48, 102175 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Tonnus, W. et al. The pathological features of regulated necrosis. J. Pathol. 247, 697–707 (2019).

    Article  CAS  PubMed  Google Scholar 

  59. Devisscher, L. et al. Discovery of novel, drug-like ferroptosis inhibitors with in vivo efficacy. J. Med. Chem. 61, 10126–10140 (2018).

    Article  CAS  PubMed  Google Scholar 

  60. Van Coillie, S. et al. Targeting ferroptosis protects against experimental (multi)organ dysfunction and death. Nat. Commun. 13, 1046 (2022).

    Article  PubMed  PubMed Central  Google Scholar 

  61. Proneth, B. & Conrad, M. Ferroptosis and necroinflammation, a yet poorly explored link. Cell Death Differ. 26, 14–24 (2019).

    Article  CAS  PubMed  Google Scholar 

  62. Drummen, G. P., van Liebergen, L. C., Op den Kamp, J. A. & Post, J. A. C11-BODIPY(581/591), an oxidation-sensitive fluorescent lipid peroxidation probe: (micro)spectroscopic characterization and validation of methodology. Free Radic. Biol. Med. 33, 473–490 (2002).

    Article  CAS  PubMed  Google Scholar 

  63. Shah, R., Farmer, L. A., Zilka, O., Van Kessel, A. T. M. & Pratt, D. A. Beyond DPPH: use of fluorescence-enabled inhibited autoxidation to predict oxidative cell death rescue. Cell Chem. Biol. 26, 1594–1607.e1597 (2019). In this study, the authors establish the FENIX assay designed to predict potential RTA activity of anti-ferroptotic compounds.

    Article  CAS  PubMed  Google Scholar 

  64. Poon, J. F., Zilka, O. & Pratt, D. A. Potent ferroptosis inhibitors can catalyze the cross-dismutation of phospholipid-derived peroxyl radicals and hydroperoxyl radicals. J. Am. Chem. Soc. 142, 14331–14342 (2020).

    Article  CAS  PubMed  Google Scholar 

  65. Zilka, O., Poon, J. F. & Pratt, D. A. Radical-trapping antioxidant activity of copper and nickel bis(thiosemicarbazone) complexes underlies their potency as inhibitors of ferroptotic cell death. J. Am. Chem. Soc. 143, 19043–19057 (2021).

    Article  CAS  PubMed  Google Scholar 

  66. Long, E. K. & Picklo, M. J. Sr Trans-4-hydroxy-2-hexenal, a product of n-3 fatty acid peroxidation: make some room HNE. Free Radic. Biol. Med. 49, 1–8 (2010).

    Article  CAS  PubMed  Google Scholar 

  67. Criscuolo, A. et al. Analytical and computational workflow for in-depth analysis of oxidized complex lipids in blood plasma. Nat. Commun. 13, 6547 (2022). This study provides a workflow based on the combination of bioinformatics and LC–MS/MS technologies to support identification and relative quantification of oxidized complex lipids.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  68. Wolk, M., Prabutzki, P. & Fedorova, M. Analytical toolbox to unlock the diversity of oxidized lipids. Acc. Chem. Res. 56, 835–845 (2023).

    Article  PubMed  Google Scholar 

  69. Ni, Z. et al. Guiding the choice of informatics software and tools for lipidomics research applications. Nat. Methods 20, 193–204 (2023).

    Article  CAS  PubMed  Google Scholar 

  70. Tyurina, Y. Y. et al. Redox phospholipidomics discovers pro-ferroptotic death signals in A375 melanoma cells in vitro and in vivo. Redox Biol. 61, 102650 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  71. Zou, Y. et al. Plasticity of ether lipids promotes ferroptosis susceptibility and evasion. Nature 585, 603–608 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  72. Cui, W., Liu, D., Gu, W. & Chu, B. Peroxisome-driven ether-linked phospholipids biosynthesis is essential for ferroptosis. Cell Death Differ. 28, 2536–2551 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  73. Perez, M. A. et al. Ether lipid deficiency disrupts lipid homeostasis leading to ferroptosis sensitivity. PLoS Genet. 18, e1010436 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  74. Breitzig, M., Bhimineni, C., Lockey, R. & Kolliputi, N. 4-Hydroxy-2-nonenal: a critical target in oxidative stress? Am. J. Physiol. Cell Physiol 311, C537–C543 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  75. Mallais, M., Hanson, C. S., Giray, M. & Pratt, D. A. General approach to identify, assess, and characterize inhibitors of lipid peroxidation and associated cell death. ACS Chem. Biol. 18, 561–571 (2023).

    Article  CAS  PubMed  Google Scholar 

  76. Vinik, Y. et al. Programming a ferroptosis-to-apoptosis transition landscape revealed ferroptosis biomarkers and repressors for cancer therapy. Adv. Sci. 11, e2307263 (2024).

    Article  Google Scholar 

  77. Feng, H. et al. Transferrin receptor is a specific ferroptosis marker. Cell Rep. 30, 3411–3423.e3417 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  78. Dixon, S. J. et al. Pharmacological inhibition of cystine-glutamate exchange induces endoplasmic reticulum stress and ferroptosis. eLife 3, e02523 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  79. Kang, Y. J., Mbonye, U. R., DeLong, C. J., Wada, M. & Smith, W. L. Regulation of intracellular cyclooxygenase levels by gene transcription and protein degradation. Prog. Lipid Res. 46, 108–125 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  80. Meinert, M. et al. Thiol starvation triggers melanoma state switching in an ATF4 and NRF2-dependent manner. Redox Biol. 70, 103011 (2024).

    Article  CAS  PubMed  Google Scholar 

  81. Oh-Hashi, K. et al. Transcriptional and post-translational regulation of mouse cation transport regulator homolog 1. Mol. Cell Biochem. 380, 97–106 (2013).

    Article  CAS  PubMed  Google Scholar 

  82. Cui, S. et al. Identification of hyperoxidized PRDX3 as a ferroptosis marker reveals ferroptotic damage in chronic liver diseases. Mol. Cell 83, 3931–3939.e3935 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  83. Hattori, K. et al. Cold stress-induced ferroptosis involves the ASK1–p38 pathway. EMBO Rep. 18, 2067–2078 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  84. Brown, K. K., Eriksson, S. E., Arner, E. S. & Hampton, M. B. Mitochondrial peroxiredoxin 3 is rapidly oxidized in cells treated with isothiocyanates. Free Radic. Biol. Med. 45, 494–502 (2008).

    Article  CAS  PubMed  Google Scholar 

  85. Takeda, K., Noguchi, T., Naguro, I. & Ichijo, H. Apoptosis signal-regulating kinase 1 in stress and immune response. Annu. Rev. Pharmacol. Toxicol. 48, 199–225 (2008).

    Article  CAS  PubMed  Google Scholar 

  86. Chorley, B. N. et al. Identification of novel NRF2-regulated genes by ChIP-Seq: influence on retinoid X receptor alpha. Nucleic Acids Res. 40, 7416–7429 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  87. Koppula, P. et al. A targetable CoQ-FSP1 axis drives ferroptosis- and radiation-resistance in KEAP1 inactive lung cancers. Nat. Commun. 13, 2206 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  88. Mishima, E. et al. DHODH inhibitors sensitize to ferroptosis by FSP1 inhibition. Nature 619, E9–E18 (2023).

    Article  CAS  PubMed  Google Scholar 

  89. Yant, L. J. et al. The selenoprotein GPX4 is essential for mouse development and protects from radiation and oxidative damage insults. Free Radic. Biol. Med. 34, 496–502 (2003).

    Article  CAS  PubMed  Google Scholar 

  90. Liang, H. et al. Short form glutathione peroxidase 4 is the essential isoform required for survival and somatic mitochondrial functions. J. Biol. Chem. 284, 30836–30844 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  91. Azuma, K. et al. Mitochondrial glutathione peroxidase 4 is indispensable for photoreceptor development and survival in mice. J. Biol. Chem. 298, 101824 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  92. Imai, H. et al. Depletion of selenoprotein GPx4 in spermatocytes causes male infertility in mice. J. Biol. Chem. 284, 32522–32532 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  93. Schneider, M. et al. Mitochondrial glutathione peroxidase 4 disruption causes male infertility. FASEB J. 23, 3233–3242 (2009).

    Article  CAS  PubMed  Google Scholar 

  94. Moreno, S. G., Laux, G., Brielmeier, M., Bornkamm, G. W. & Conrad, M. Testis-specific expression of the nuclear form of phospholipid hydroperoxide glutathione peroxidase (PHGPx). Biol. Chem. 384, 635–643 (2003).

    Article  CAS  PubMed  Google Scholar 

  95. Conrad, M. et al. The nuclear form of phospholipid hydroperoxide glutathione peroxidase is a protein thiol peroxidase contributing to sperm chromatin stability. Mol. Cell Biol. 25, 7637–7644 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  96. Labunskyy, V. M., Hatfield, D. L. & Gladyshev, V. N. Selenoproteins: molecular pathways and physiological roles. Physiol. Rev. 94, 739–777 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  97. Li, Z. et al. Ribosome stalling during selenoprotein translation exposes a ferroptosis vulnerability. Nat. Chem. Biol. 18, 751–761 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  98. Alborzinia, H. et al. LRP8-mediated selenocysteine uptake is a targetable vulnerability in MYCN-amplified neuroblastoma. EMBO Mol. Med. 15, e18014 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  99. Nakamura, T. et al. A tangible method to assess native ferroptosis suppressor activity. Cell Rep. Methods 4, 100710 (2024).

    Article  PubMed  PubMed Central  Google Scholar 

  100. Hurst, R. et al. Hyperresistance to cholesterol hydroperoxide-induced peroxidative injury and apoptotic death in a tumor cell line that overexpresses glutathione peroxidase isotype-4. Free Radic. Biol. Med. 31, 1051–1065 (2001).

    Article  CAS  PubMed  Google Scholar 

  101. Vuckovic, A. M. et al. Inactivation of the glutathione peroxidase GPx4 by the ferroptosis-inducing molecule RSL3 requires the adaptor protein 14-3-3epsilon. FEBS Lett. 594, 611–624 (2020).

    Article  CAS  PubMed  Google Scholar 

  102. Sato, H., Tamba, M., Kuriyama-Matsumura, K., Okuno, S. & Bannai, S. Molecular cloning and expression of human xCT, the light chain of amino acid transport system xc. Antioxid. Redox Signal. 2, 665–671 (2000).

    Article  CAS  PubMed  Google Scholar 

  103. Sato, H. et al. Redox imbalance in cystine/glutamate transporter-deficient mice. J. Biol. Chem. 280, 37423–37429 (2005).

    Article  CAS  PubMed  Google Scholar 

  104. Armenta, D. A. et al. Ferroptosis inhibition by lysosome-dependent catabolism of extracellular protein. Cell Chem. Biol. 29, 1588–1600.e1587 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  105. Zheng, J. et al. Sorafenib fails to trigger ferroptosis across a wide range of cancer cell lines. Cell Death Dis. 12, 698 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  106. Poltorack, C. D. & Dixon, S. J. Understanding the role of cysteine in ferroptosis: progress & paradoxes. FEBS J. 289, 374–385 (2022).

    Article  CAS  PubMed  Google Scholar 

  107. Kobayashi, S. et al. Carnosine dipeptidase II (CNDP2) protects cells under cysteine insufficiency by hydrolyzing glutathione-related peptides. Free Radic. Biol. Med. 174, 12–27 (2021).

    Article  CAS  PubMed  Google Scholar 

  108. Lee, J. et al. The viability of primary hepatocytes is maintained under a low cysteine-glutathione redox state with a marked elevation in ophthalmic acid production. Exp. Cell Res. 361, 178–191 (2017).

    Article  CAS  PubMed  Google Scholar 

  109. Van Liefferinge, J. et al. Comparative analysis of antibodies to xCT (Slc7a11): forewarned is forearmed. J. Comp. Neurol. 524, 1015–1032 (2016).

    Article  PubMed  Google Scholar 

  110. Nguyen, H. P. et al. Aifm2, a NADH oxidase, supports robust glycolysis and is required for cold- and diet-induced thermogenesis. Mol. Cell 77, 600–617.e604 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  111. Mishima, E., Wahida, A., Seibt, T. & Conrad, M. Diverse biological functions of vitamin K: from coagulation to ferroptosis. Nat. Metab. 5, 924–932 (2023).

    Article  CAS  PubMed  Google Scholar 

  112. Tonnus, W. et al. Dysfunction of the key ferroptosis-surveilling systems hypersensitizes mice to tubular necrosis during acute kidney injury. Nat. Commun. 12, 4402 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  113. Soriano-Castell, D., Liang, Z., Maher, P. & Currais, A. Profiling the chemical nature of anti-oxytotic/ferroptotic compounds with phenotypic screening. Free Radic. Biol. Med. 177, 313–325 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  114. Nakamura, T. et al. Integrated chemical and genetic screens unveil FSP1 mechanisms of ferroptosis regulation. Nat. Struct. Mol. Biol. 30, 1806–1815 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  115. Xavier da Silva, T. N., Schulte, C., Alves, A. N., Maric, H. M. & Friedmann Angeli, J. P. Molecular characterization of AIFM2/FSP1 inhibition by iFSP1-like molecules. Cell Death Dis. 14, 281 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  116. Magtanong, L. et al. Context-dependent regulation of ferroptosis sensitivity. Cell Chem. Biol. 29, 1409–1418.e1406 (2022).

    Article  CAS  PubMed  Google Scholar 

  117. Shimbara-Matsubayashi, S., Kuwata, H., Tanaka, N., Kato, M. & Hara, S. Analysis on the substrate specificity of recombinant human acyl-CoA synthetase ACSL4 variants. Biol. Pharm. Bull. 42, 850–855 (2019).

    Article  CAS  PubMed  Google Scholar 

  118. Cao, Y., Traer, E., Zimmerman, G. A., McIntyre, T. M. & Prescott, S. M. Cloning, expression, and chromosomal localization of human long-chain fatty acid-CoA ligase 4 (FACL4). Genomics 49, 327–330 (1998).

    Article  CAS  PubMed  Google Scholar 

  119. Piccini, M. et al. FACL4, a new gene encoding long-chain acyl-CoA synthetase 4, is deleted in a family with Alport syndrome, elliptocytosis, and mental retardation. Genomics 47, 350–358 (1998).

    Article  CAS  PubMed  Google Scholar 

  120. Klett, E. L., Chen, S., Yechoor, A., Lih, F. B. & Coleman, R. A. Long-chain acyl-CoA synthetase isoforms differ in preferences for eicosanoid species and long-chain fatty acids. J. Lipid Res. 58, 884–894 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  121. Chen, W. C. et al. Systematic analysis of gene expression alterations and clinical outcomes for long-chain acyl-coenzyme A synthetase family in cancer. PLoS ONE 11, e0155660 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  122. Zilka, O. et al. On the mechanism of cytoprotection by ferrostatin-1 and liproxstatin-1 and the role of lipid peroxidation in ferroptotic cell death. ACS Cent. Sci. 3, 232–243 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  123. Helberg, J. & Pratt, D. A. Autoxidation vs. antioxidants — the fight for forever. Chem. Soc. Rev. 50, 7343–7358 (2021).

    Article  CAS  PubMed  Google Scholar 

  124. Mishima, E. et al. Drugs repurposed as antiferroptosis agents suppress organ damage, including AKI, by functioning as lipid peroxyl radical scavengers. J. Am. Soc. Nephrol. 31, 280–296 (2020).

    Article  CAS  PubMed  Google Scholar 

  125. Conlon, M. et al. A compendium of kinetic modulatory profiles identifies ferroptosis regulators. Nat. Chem. Biol. 17, 665–674 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  126. Shah, R., Shchepinov, M. S. & Pratt, D. A. Resolving the role of lipoxygenases in the initiation and execution of ferroptosis. ACS Cent. Sci. 4, 387–396 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  127. Sato, H. et al. Transcriptional control of cystine/glutamate transporter gene by amino acid deprivation. Biochem. Biophys. Res. Commun. 325, 109–116 (2004).

    Article  CAS  PubMed  Google Scholar 

  128. Forcina, G. C. et al. Ferroptosis regulation by the NGLY1/NFE2L1 pathway. Proc. Natl Acad. Sci. USA 119, e2118646119 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  129. Chen, Y. et al. Quantitative profiling of protein carbonylations in ferroptosis by an aniline-derived probe. J. Am. Chem. Soc. 140, 4712–4720 (2018).

    Article  CAS  PubMed  Google Scholar 

  130. Gao, J. et al. Selenium-encoded isotopic signature targeted profiling. ACS Cent. Sci. 4, 960–970 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  131. Eaton, J. K. et al. Selective covalent targeting of GPX4 using masked nitrile-oxide electrophiles. Nat. Chem. Biol. 16, 497–506 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  132. Chen, T. et al. Discovery of novel potent covalent glutathione peroxidase 4 inhibitors as highly selective ferroptosis inducers for the treatment of triple-negative breast cancer. J. Med. Chem. 66, 10036–10059 (2023).

    Article  CAS  PubMed  Google Scholar 

  133. Yan, B. et al. Membrane damage during ferroptosis is caused by oxidation of phospholipids catalyzed by the oxidoreductases POR and CYB5R1. Mol. Cell 81, 355–369.e310 (2021).

    Article  CAS  PubMed  Google Scholar 

  134. Oh, M. et al. The lipoprotein-associated phospholipase A2 inhibitor Darapladib sensitises cancer cells to ferroptosis by remodelling lipid metabolism. Nat. Commun. 14, 5728 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  135. Li, J. et al. Tumor-specific GPX4 degradation enhances ferroptosis-initiated antitumor immune response in mouse models of pancreatic cancer. Sci. Transl. Med. 15, eadg3049 (2023).

    Article  CAS  PubMed  Google Scholar 

  136. Luo, T. et al. Intracellular delivery of glutathione peroxidase degrader induces ferroptosis in vivo. Angew. Chem. Int. Ed. Engl. 61, e202206277 (2022).

    Article  CAS  PubMed  Google Scholar 

  137. Cheff, D. M. et al. The ferroptosis inducing compounds RSL3 and ML162 are not direct inhibitors of GPX4 but of TXNRD1. Redox Biol. 62, 102703 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  138. Fujii, J., Homma, T. & Kobayashi, S. Ferroptosis caused by cysteine insufficiency and oxidative insult. Free Radic. Res. 54, 969–980 (2020).

    Article  CAS  PubMed  Google Scholar 

  139. Sagara, J. I., Miura, K. & Bannai, S. Maintenance of neuronal glutathione by glial cells. J. Neurochem. 61, 1672–1676 (1993).

    Article  CAS  PubMed  Google Scholar 

  140. Gao, M. et al. Role of mitochondria in ferroptosis. Mol. Cell 73, 354–363.e353 (2019).

    Article  CAS  PubMed  Google Scholar 

  141. Zhang, Y. et al. Imidazole ketone erastin induces ferroptosis and slows tumor growth in a mouse lymphoma model. Cell Chem. Biol. 26, 623–633.e629 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  142. Gout, P. W., Buckley, A. R., Simms, C. R. & Bruchovsky, N. Sulfasalazine, a potent suppressor of lymphoma growth by inhibition of the xc− cystine transporter: a new action for an old drug. Leukemia 15, 1633–1640 (2001).

    Article  CAS  PubMed  Google Scholar 

  143. Griffith, O. W. & Meister, A. Potent and specific inhibition of glutathione synthesis by buthionine sulfoximine (S-n-butyl homocysteine sulfoximine). J. Biol. Chem. 254, 7558–7560 (1979).

    Article  CAS  PubMed  Google Scholar 

  144. Harris, I. S. et al. Deubiquitinases maintain protein homeostasis and survival of cancer cells upon glutathione depletion. Cell Metab. 29, 1166–1181.e1166 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  145. Xia, C. et al. Cysteine and homocysteine can be exploited by GPX4 in ferroptosis inhibition independent of GSH synthesis. Redox Biol. 69, 102999 (2024).

    Article  CAS  PubMed  Google Scholar 

  146. Bebber, C. M. et al. Ferroptosis response segregates small cell lung cancer (SCLC) neuroendocrine subtypes. Nat. Commun. 12, 2048 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  147. Harris, I. S. et al. Glutathione and thioredoxin antioxidant pathways synergize to drive cancer initiation and progression. Cancer Cell 27, 211–222 (2015).

    Article  CAS  PubMed  Google Scholar 

  148. Fujihara, K. M. et al. Eprenetapopt triggers ferroptosis, inhibits NFS1 cysteine desulfurase, and synergizes with serine and glycine dietary restriction. Sci. Adv. 8, eabm9427 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  149. Nakamura, T. et al. Phase separation of FSP1 promotes ferroptosis. Nature 619, 371–377 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  150. Hendricks, J. M. et al. Identification of structurally diverse FSP1 inhibitors that sensitize cancer cells to ferroptosis. Cell Chem. Biol. 30, 1090–1103.e1097 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  151. Falk, M. H., Hultner, L., Milner, A., Gregory, C. D. & Bornkamm, G. W. Irradiated fibroblasts protect Burkitt lymphoma cells from apoptosis by a mechanism independent of BCL-2. Int. J. Cancer 55, 485–491 (1993).

    Article  CAS  PubMed  Google Scholar 

  152. Takashima, H. et al. Impact of selenium content in fetal bovine serum on ferroptosis susceptibility and selenoprotein expression in cultured cells. J. Toxicol. Sci. 49, 555–563 (2024).

    Article  CAS  PubMed  Google Scholar 

  153. Mishima, E. & Conrad, M. Nutritional and metabolic control of ferroptosis. Annu. Rev. Nutr. 42, 275–309 (2022).

    Article  CAS  PubMed  Google Scholar 

  154. Gao, M., Monian, P., Quadri, N., Ramasamy, R. & Jiang, X. Glutaminolysis and transferrin regulate ferroptosis. Mol. Cell 59, 298–308 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  155. Lee, H. et al. Energy-stress-mediated AMPK activation inhibits ferroptosis. Nat. Cell Biol. 22, 225–234 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  156. Vuckovic, A. M. et al. Aerobic pyruvate metabolism sensitizes cells to ferroptosis primed by GSH depletion. Free Radic. Biol. Med. 167, 45–53 (2021).

    Article  CAS  PubMed  Google Scholar 

  157. Song, X. et al. PDK4 dictates metabolic resistance to ferroptosis by suppressing pyruvate oxidation and fatty acid synthesis. Cell Rep. 34, 108767 (2021).

    Article  CAS  PubMed  Google Scholar 

  158. Vera, M., Barahona, M. J., Nova-Lamperti, E., Nualart, F. & Ferrada, L. The phenol red compound: a potential artifact in pharmacological induction of ferroptosis. Free Radic. Biol. Med. 222, 397–402 (2024).

    Article  CAS  PubMed  Google Scholar 

  159. Deshwal, S. et al. Mitochondria regulate intracellular coenzyme Q transport and ferroptotic resistance via STARD7. Nat. Cell Biol. 25, 246–257 (2023).

    CAS  PubMed  PubMed Central  Google Scholar 

  160. Imai, H. et al. Early embryonic lethality caused by targeted disruption of the mouse PHGPx gene. Biochem. Biophys. Res. Commun. 305, 278–286 (2003).

    Article  CAS  PubMed  Google Scholar 

  161. Conrad, M., Lorenz, S. M. & Proneth, B. Targeting ferroptosis: new hope for as-yet-incurable diseases. Trends Mol. Med. 27, 113–122 (2021).

    Article  CAS  PubMed  Google Scholar 

  162. Chen, L., Hambright, W. S., Na, R. & Ran, Q. Ablation of the ferroptosis inhibitor glutathione peroxidase 4 in neurons results in rapid motor neuron degeneration and paralysis. J. Biol. Chem. 290, 28097–28106 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  163. Wirth, E. K. et al. Neuronal selenoprotein expression is required for interneuron development and prevents seizures and neurodegeneration. FASEB J. 24, 844–852 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  164. Carlson, B. A. et al. Glutathione peroxidase 4 and vitamin E cooperatively prevent hepatocellular degeneration. Redox Biol. 9, 22–31 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  165. Wortmann, M. et al. Combined deficiency in glutathione peroxidase 4 and vitamin E causes multiorgan thrombus formation and early death in mice. Circ. Res. 113, 408–417 (2013).

    Article  CAS  PubMed  Google Scholar 

  166. Matsushita, M. et al. T cell lipid peroxidation induces ferroptosis and prevents immunity to infection. J. Exp. Med. 212, 555–568 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  167. Alim, I. et al. Selenium drives a transcriptional adaptive program to block ferroptosis and treat stroke. Cell 177, 1262–1279.e1225 (2019).

    Article  CAS  PubMed  Google Scholar 

  168. Yamada, N. et al. Ferroptosis driven by radical oxidation of n-6 polyunsaturated fatty acids mediates acetaminophen-induced acute liver failure. Cell Death Dis. 11, 144 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  169. Martin-Sanchez, D. et al. Ferroptosis, but not necroptosis, is important in nephrotoxic folic acid-induced AKI. J. Am. Soc. Nephrol. 28, 218–229 (2017).

    Article  CAS  PubMed  Google Scholar 

  170. Van San, E. et al. Ferroptosis contributes to multiple sclerosis and its pharmacological targeting suppresses experimental disease progression. Cell Death Differ. 30, 2092–2103 (2023).

    Article  PubMed  PubMed Central  Google Scholar 

  171. Ide, S. et al. Sex differences in resilience to ferroptosis underlie sexual dimorphism in kidney injury and repair. Cell Rep. 41, 111610 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  172. Ward, N. P., Kang, Y. P., Falzone, A., Boyle, T. A. & DeNicola, G. M. Nicotinamide nucleotide transhydrogenase regulates mitochondrial metabolism in NSCLC through maintenance of Fe-S protein function. J. Exp. Med. 217, e20191689 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  173. Co, H. K. C., Wu, C. C., Lee, Y. C. & Chen, S. H. Emergence of large-scale cell death through ferroptotic trigger waves. Nature 631, 654–662 (2024).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  174. Davidson, A. J., Heron, R., Das, J., Overholtzer, M. & Wood, W. Ferroptosis-like cell death promotes and prolongs inflammation in Drosophila. Nat. Cell Biol. 26, 1535–1544 (2024).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  175. Ni, Z. et al. Evaluation of air oxidized PAPC: a multi laboratory study by LC–MS/MS. Free Radic. Biol. Med. 144, 156–166 (2019).

    Article  CAS  PubMed  Google Scholar 

  176. Criscuolo, A., Zeller, M. & Fedorova, M. Evaluation of lipid in-source fragmentation on different orbitrap-based mass spectrometers. J. Am. Soc. Mass. Spectrom. 31, 463–466 (2020).

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

We thank all our colleagues, especially K. Ono, J. Ito, W. Zhang, A. Levkina, M. Novikova, J. Zheng, T. Seibt, M. Aldrovandi, S.M. Lorenz, D. Chen, N. Yamada, G. Mardani, C. Xu and B. Henkelmann, for their help in organizing this Expert Recommendation. We thank S. Kobayashi and H. Sato for providing expert views about system xc−. M.C. received funding from the Deutsche Forschungsgemeinschaft (CO 291/7-1, the Priority Program SPP 2306 (grants CO 291/9-1, 461385412; CO 291/10-1, 461507177, CO 291/9-2, CO 291/10-2, CO 291/14-1) and the CRC TRR 353 (CO 291/11-1; 471011418), the German Federal Ministry of Education and Research FERROPATH (grant 01EJ2205B) and the European Research Council under the European Union’s Horizon 2020 research and innovation program (grant GA 884754). D.A.P. thanks the Natural Sciences and Engineering Research Council of Canada for their continued support (RGPIN-2022-05058). Work in the Fedorova lab is supported by ‘Sonderzuweisung zur Unterstützung profilbestimmender Struktureinheiten der TUD’ by the SMWK, TG70 by Sächsische Aufbaubank and SMWK, the measure is cofinanced with tax funds based on the budget passed by the Saxon state parliament (to M.F.), Deutsche Forschungsgemeinschaft (grant FE 1236/5-1 to M.F.) and Bundesministerium für Bildung und Forschung (grant 01EJ2205A, FERROPath to M.F.). S.J.D. is supported by the USA National Institutes of Health (grant R01GM122923). E.M. thanks the Research Grant of Sapporo Bioscience Foundation and Food Science Institute Foundation (Ryoshoku Kenkyuukai).

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Author notes

  1. These authors contributed equally: Eikan Mishima, Toshitaka Nakamura, Adam Wahida, Marcus Conrad.

Authors and Affiliations

  1. Institute of Metabolism and Cell Death, Molecular Targets and Therapeutics Center, Helmholtz Munich, Neuherberg, Germany

    Eikan Mishima, Toshitaka Nakamura, Sebastian Doll, Bettina Proneth, Adam Wahida & Marcus Conrad

  2. Department of Nephrology, Tohoku University Graduate School of Medicine, Sendai, Japan

    Eikan Mishima

  3. Center of Membrane Biochemistry and Lipid Research, University Hospital Carl Gustav Carus and Faculty of Medicine of TU Dresden, Dresden, Germany

    Maria Fedorova

  4. Department of Chemistry and Biomolecular Science, University of Ottawa, Ottawa, Ontario, Canada

    Derek A. Pratt

  5. Rudolf Virchow Center for Integrative and Translational Bioimaging, University of Würzburg, Würzburg, Germany

    José Pedro Friedmann Angeli

  6. Department of Biology, Stanford University, Stanford, CA, USA

    Scott J. Dixon

  7. Translational Redox Biology, TUM Natural School of Sciences, Technical University of Munich, Garching, Germany

    Marcus Conrad

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Contributions

E.M., T.N., A.W. and M.C. wrote the article. S.D., B.P., M.F., D.A.P., J.P.F.A. and S.J.D. contributed substantially to the discussion of the content. All authors reviewed and edited the manuscript before submission.

Corresponding author

Correspondence to Marcus Conrad.

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Competing interests

M.C. and B.P. are cofounders and shareholders of ROSCUE Therapeutics GmbH. D.A.P. is a cofounder and shareholder of Prothegen Inc. S.J.D. holds patents related to ferroptosis. M.C., B.P. and T.N. have filed patents for some of the compounds described herein.

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41580_2025_843_MOESM2_ESM.mp4

Supplementary Video 1. Live imaging of cells undergoing ferroptosis. Pfa1 cells with inducible Gpx4 deletion following treatment with 4-hydroxytamoxifen. The video was captured using Nanolive 3D Cell Explorer 42–56 h after the addition of 4-hydroxytamoxifen. E.M. produced the video.

Glossary

3′-UTR selenocysteine insertion sequence element

A stem loop-like secondary structure located in the 3′-untranslated region (UTR) of selenoprotein mRNAs, which affords decoding of UGA as a selenocysteine during translation.

4-hydroxy-2-nonenal

(4-HNE). An α,β-unsaturated hydroxyalkenal produced downstream of lipid peroxidation, which can react with specific amino acid residues (that is, histidine, cysteine and lysine) in proteins to generate Michael adducts in cells and tissues.

C57BL/6J × 129S6/SvEv mixed background

F1 mice with mixed background strains derived from two inbred strains of C57BL/6J and 129S6/SvEv mice.

Epilipidomic studies

A method to analyse a subset of natural lipidome formed by lipid modifications (for example oxidation) required to regulate complex biological functions.

Fenton reaction

A reaction in which iron or other transition metals catalyse the disproportionation of hydrogen peroxide into highly reactive hydroxyl radical and a hydroperoxide ion.

Gpx4 U46C mutant mice

Transgenic mice carrying a targeted mutation of the catalytically active site selenocysteine (U46) to Cys of Gpx4.

Ischaemia-reperfusion injury

(IRI). Transient ischaemia, followed by reperfusion, generates oxygen-centred radicals that trigger extensive cell death and inflammatory responses in the affected organs, leading to acute tissue damage.

Karyolysis

Complete dissolution of nuclear components in a dying cell.

Lipid hydroperoxides

The primary products of lipid peroxidation, resulting from propagation by H-atom transfer, can be further reduced or oxidized to yield radicals that initiate additional lipid peroxidation and/or produce secondary reactive lipid aldehydes.

Lipid peroxidation

Generally refers to the autoxidation of lipids, a free radical chain reaction in which oxygen is incorporated into hydrocarbons to form peroxides, resulting in the production of lipid hydroperoxides when the chain reaction is propagated by H-atom transfer from a lipid or lipid peroxides when propagated by addition to a lipid.

Necroptosis

A regulated, necrotic cell death modality mediated by receptor-interacting protein kinase 3 (RIPK3) activity and ensuing pore formation by mixed-lineage kinase domain-like pseudokinase.

Parvalbumin-expressing GABAergic interneurons

The principal inhibitory interneurons in the brain cortex.

Phospholipids

Amphiphilic molecules with a hydrophilic head containing a phosphate group (for example phosphocholine and phosphoethanolamine) and two hydrophobic fatty acid tails esterified to the glycerol moiety, which are key component of cell membranes.

Plasmalogens

A unique class of phospholipids containing a vinyl ether bond at the sn-1 position, with its synthesis initiated in peroxisomes and endoplasmic reticulum.

Pyknosis

Condensation of the nucleus and chromatin, often observed in cells undergoing cell death.

Radical-trapping antioxidants

(RTAs). Compounds that react with radical chain-propagating radicals to form non-propagating radicals31,123.

Regulated necrosis

A type of programmed cell death involving plasma membrane rupture and including various modalities, such as ferroptosis and necroptosis.

Selenocysteinyl-tRNA

A specific tRNA responsible for incorporating selenocysteine into selenoproteins during translation.

Selenoproteins

An exclusive group of selenium-containing proteins in which selenocysteine, the 21st proteinogenic amino acid, is cotranslationally incorporated into the protein and is usually present at the catalytically active site.

Tetrahydrobiopterin

(BH4). A redox-active cofactor for several biosynthetic enzymes, also functioning as an RTA by reacting with peroxyl radicals, yielding oxidation products such as dihydrobiopterin (BH2), which can be reduced back to BH4 by the enzyme dihydrofolate reductase.

Thiol-containing molecules

Organic molecules that contain a sulfhydryl group.

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Mishima, E., Nakamura, T., Doll, S. et al. Recommendations for robust and reproducible research on ferroptosis. Nat Rev Mol Cell Biol 26, 615–630 (2025). https://doi.org/10.1038/s41580-025-00843-2

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