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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. 4 — a, Immunoblots showing the expression of ACSL4 and p-ACSL4(T679) in PCK2-knockout cells and parental cells. b, Immunoblots showing the expression of ACSL4 and p-ACSL4(T679) in TRCs and bulk tumor cells. c, Representative immunofluorescence staining image showing the co-localization of ACSL4 and mitochondria in cells stably transfected with mito-GFP. Scale bar, 10 μm. d, Immunoblots showing the localization of ACSL4 in mitochondria. Cyto, cytosol; Mito, mitochondria; WCL, whole-cell lysate. Tom20 and α-tubulin are markers for mitochondria and cytosol, respectively. e, Immunoelectron microscopic localization of ACSL4 and PCK2 in the mitochondria by using a gold-labeled anti-ACSL4 antibody (15 nm gold) and a gold-labeled anti-PCK2 antibody (6 nm gold). The co-localization of ACSL4 and PCK2 in sections of HONE1 cells is shown. Bars, 0.2 μm. f, Immunoprecipitation and immunoblotting showing the interaction of ACSL4 and PCK2 in HONE1 TRCs and bulk cells. g, PCK2-knockout HONE1 cells and parental cells were treated with AA-d8 (10 µM). The relative changes of PE that contain AA (20:4)-d8 are shown. n = 4. *P < 0.05; **P < 0.01; ***P < 0.001. P = 0.0254, 0.0294, 0.0038, 0.0024, 0.0027, 0.0267, 0.0225 and 0.004. h, Accumulation of RSL3-induced oxPE that contains AA or AdA in PCK2-knockout HONE1 cells and parental cells. P = 0.0000417, 0.0039 and 0.0046. i, Dose-dependent toxicity of RSL3-induced cell death of PCK2-knockout HONE1 cells (sg PCK2) and parental cells (sg GFP). j, Parental HONE1 cells and TRCs from PCK2-knockout HONE1 cells stably expressing Dox-inducible PCK2 (PCK2^Tet) were treated without or with Dox. Immunoblots showing the expression of p-ACSL4(T679) and ACSL4 in cells. k, The cells depicted in j were treated with or without Dox before RSL3 or FINO2 treatment (15 μM). The percentage of dead cells was measured by 7-AAD staining and flow cytometry. Data are shown as mean ± s.d. (g,h,k); unpaired two-tailed t-test (g) or one-way ANOVA (h,k). n = 3 independent experiments. One of three experiments is shown (a,b,d,f,j).

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