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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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Fig. 3 — a, Immunoprecipitation and immunoblot analysis of the level of phospho-Ser/Thr ACSL4 in TRCs and bulk tumor cells (HONE1 and A375). b, Mass spectrometry analysis of the phosphorylation site in ACSL4 protein purified from HONE1 TRCs or bulk cells (left). The percentage of ACSL4 Thr679 phosphorylation in HONE1 TRCs and bulk tumor cells (right). c, The formation rate of AA-d8-CoA in reactions catalyzed by ACSL4 protein purified from Dox-inducible ACSL4-WT HONE1 cells or Dox-inducible ACSL4-T679A HONE1 cells. AA-d8 and coenzyme A were used as substrates. AA-d8-CoA was undetectable in the samples with no enzyme added. d, Dose-dependent toxicity of RSL3-induced cell death of ACSL4-knockout HONE1 cells stably expressing Dox-inducible ACSL4-WT (ACSL4 WT^Tet) or Dox-inducible ACSL4-T679A (ACSL4 T679A^Tet) with or without Dox (0.3 μg ml⁻¹). e, The radar chart indicates the changes of SFA-PLs, MUFA-PLs and PUFA-PLs in Dox-inducible ACSL4-WT HONE1 cells or Dox-inducible ACSL4-T679A HONE1 cells (left). The relative increment of these PLs is represented by a bar chart (right). f,g, Dox-inducible ACSL4-WT HONE1 cells or Dox-inducible ACSL4-T679A HONE1 cells were treated with Dox (0.3 μg ml⁻¹) followed by AA-d8 (10 µM) for 36 h. The relative changes of PC (f) or PE (g) that contain AA (20:4)-d8 are shown. n = 4. *P < 0.05; **P < 0.01; ***P < 0.001. f, P = 0.0017, 0.0008, 0.0236, 0.0017, 0.0045, 0.0035, 0.0373, 0.0312, 0.0013 and 0.0030. g, P = 0.0481, 0.0039, 0.0263, 0.0045, 0.0446, 0.0164, 0.0073, 0.0035 and 0.0002. h, Accumulation of RSL3-induced oxygenated PE that contains AA or AdA 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⁻¹). P = 0.000000182, 0.0004 and 0.0000171. Data are shown as mean ± s.d.; unpaired two-tailed t-test (c,f,g) or one-way ANOVA (h). n = 3 independent experiments. One of three experiments is shown (a).

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