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. 2024 May 8;20(10):1341–1352. doi: 10.1038/s41589-024-01612-6

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© The Author(s) 2024

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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Extended Data Fig. 4 — a, Alignment of protein sequences spanning ACSL4 T679 from different species. b, Immunoblots showing the ACSL4 expression in ACSL4-knockout HONE1 cells transfected with Dox-inducible ACSL4-T679A mutation expression plasmids. Cells were treated without or with Dox. c, Dose-dependent toxicity of FINO2 induced cell death of ACSL4- knockout HONE1 cells stably expressing Doxycycline (Dox)- inducible ACSL4-wild type (ACSL4 WT^Tet) or Dox-inducible ACSL4-T679A (ACSL4 T679A^Tet) without or with Dox. d, Dox-inducible ACSL4-WT HONE1 cells or Dox-inducible ACSL4-T679A HONE1 cells pretreated with Dox, followed by IR or cisplatin. The percentage of dead cells was measured. P = 0.0000104 and 0.0005. e, Principal component analysis (PCA) of RSL3-induced oxygenated phospholipids in ACSL4-knockout HONE1 cells (sg ACSL4), Dox-inducible ACSL4-WT expression HONE1 cells (ACSL4 WT^Tet) or Dox-inducible ACSL4-T679A expression HONE1 cells (ACSL4 T679A^Tet) treated with Dox, with or without RSL3. Lipids were analyzed by LC-MS/MS. Each point represents a sample. f, Heatmap showing LC-MS analysis of the relative changes in RSL3-induced oxygenated PE molecular species in ACSL4-knockout HONE1 cells (sg ACSL4), Dox-inducible ACSL4-WT expression HONE1 cells (ACSL4 WT^Tet) or Dox-inducible ACSL4-T679A expression HONE1 cells (ACSL4 T679A^Tet) treated with Dox (0.3 μg/mL). Row z-scores were obtained from averaged values of the content of lipid species. g, Microscale thermophoresis (MST) demonstrated a direct interaction between Arachidonic acid and GFP-tagged ACSL4 in lysates from GFP-ACSL4-WT or GFP-ACSL4-T679A expressing HEK293T cells; mutation of T679A partly blocks the interaction. Data are shown as mean ± SD, one-way ANOVA (d), n = 3 independent experiments. One of three experiments is shown (b, g). aa, amino acid.

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