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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. 3 — a, Immunoblots showing the expression of ACSL4 and LPCAT3 in TRCs and bulk tumor cells. b, Cell lysates of TRCs and bulk tumor cells from HONE1 cells were incubated with or without LPCAT3 inhibitors, (R)-HTS-3. LPCAT3 enzymatic activity was assessed. c, Cell lysates of TRCs and bulk tumor cells from HONE1 cells were incubated with or without ACSL4 inhibitors, Rosiglitazone. The formation of AA-d8-CoA in reactions was detected by LC-MS. d, Immunoblots showing the expression of ACSL4 in ACSL4-knockout HONE1 cells and A375 cells. e, TRCs and bulk cells from ACSL4-knockout A375 cells or parental cells were treated with RSL3 or FINO2. The percentage of dead cells was measured. f-g, Percentage of dead cells in TRCs and bulk tumor cells from HONE1 cells (f) or A375 cells (g) with indicated treatment. Rosiglitazone (ROSI, 10 µM) was added before the treatment of ferroptosis inducers. h, Immunoblots showing the expression of ACSL4 in ACSL4- knockout HONE1 cells transfected with Dox-inducible ACSL4-WT expression plasmids. Cells were treated with or without Dox. i, The parental HONE1 cells (sg GFP), TRCs and bulk tumor cells from ACSL4-knockout HONE1 cells (sg ACSL4) and ACSL4-knockout HONE1 cells stably expressing Dox-inducible ACSL4-wild type (ACSL4 WT^Tet) were treated with Dox before RSL3 treatment. The percentage of dead cells was measured by 7-AAD staining and flow cytometry. P = 0.8154, 0.0000173 and 0.000000000998. j, Immunoblots showing the expression of ACSL4 in the cells depicted in (i). k-l, The parental HONE1 cells (sg GFP), TRCs and bulk tumor cells from ACSL4-knockout HONE1 cells (sg ACSL4) and ACSL4- knockout HONE1 cells stably expressing Dox-inducible ACSL4-wild type (ACSL4 WT^Tet) were treated with or without Dox, followed by treatment with IR (k) or cisplatin (Cis) (l). The percentage of dead cells was measured. P = 0.0951, 0.0000000432, 0.6187 and 0.00000273. m, Immunoblots showing the expression of ACSL4 in the cells depicted in (k-l). Data are shown as mean ± SD, unpaired two-tailed t-test (b-c, e) or one-way ANOVA (i, k-l), n = 3 independent experiments. One of three experiments is shown (a, d, h, j, m). ns, not significant.

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