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. 2021 Oct 27;12(11):1009. doi: 10.1038/s41419-021-04284-5

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© The Author(s) 2021

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 license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license 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 license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 3 — Keratinocytes were stimulated with 10 μM erastin in the absence or presence of the indicated inhibitor 0.5 μM Fer-1 for 24 h. A The CCK8 assay shows the viability of keratinocytes. B Western blot for GPX4, 4-HNE-modified protein levels, and ACSL4. Actin was used as the loading control. C GSH/GSSG changes seen in keratinocytes. D MDA level observed in keratinocytes. E Production of ROS observed by flow cytometry (left). Production of ROS was analyzed, and data were expressed as arbitrary units of DCF fluorescence (Right). F Generation of lipid hydroperoxides observed by flow cytometry (left). Levels of lipid hydroperoxides were expressed as BODIPY-C11Oxidized/ (BODIPY-C11Oxidized + BODIPY-C11Non-oxidized) ratio (Right). G Live-cell fluorescence imaging of lipid hydroperoxides in keratinocytes. Oxidized lipid is in green, a non-oxidized lipid is represented in orange. Scale bar, 20 μm. Values were presented as the mean ± standard error (n = 5). *P < 0.05,**P < 0.01,***P < 0.001. Independent experiments were repeated three times. ACSL4 acyl-CoA synthetase long-chain family member 4, GPX4 glutathione peroxidase 4, 4-HNE 4-hydroxynonenal, Fer-1 ferrostatin-1, MDA malondialdehyde, GSH glutathione, GSSG oxidized glutathione, ROS reactive oxygen species.