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. 2024 Feb 23;43(7):1164–1186. doi: 10.1038/s44318-024-00055-y

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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/. Creative Commons Public Domain Dedication waiver http://creativecommons.org/publicdomain/zero/1.0/ applies to the data associated with this article, unless otherwise stated in a credit line to the data, but does not extend to the graphical or creative elements of illustrations, charts, or figures. This waiver removes legal barriers to the re-use and mining of research data. According to standard scholarly practice, it is recommended to provide appropriate citation and attribution whenever technically possible.

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(A–D) Percentage of propidium iodide (PI, Mw = 668 Da) uptake in WT and Ninj1^−/− BMDMs over time (0–8 h) after treatment with 5μg/mL nigericin (A), 1 mM CuOOH (B), 5 μM RSL3 (C), or 5 μM ML162 (D). When indicated, 25 μM Fer-1 was added simultaneously with ferroptosis activators. (E–G) Release of 3 kDa fluorescent dextran from WT and Ninj1^−/− BMDMs left untreated (UT) or treated with 1 mM CuOOH (E), 5 μM RSL3 (F), or 5 μM ML162 (G) over time (0–8 h). (H) Time-lapse fluorescence confocal microscopy of HeLa cells expressing hNINJ1-GFP after 1 mM CuOOH treatment. Images show the green fluorescence at the basal plane of the cell and the influx of DRAQ7 (maximum projection from a Z-stack) to track plasma membrane permeabilization. White arrows indicate regions that are enlarged in the insets. Time was normalized to the onset of increase in DRAQ7 nuclear fluorescence. Scale bar: 20 µm. (I, J) Normalized quantification of the inhomogeneity distribution of NINJ1-GFP (I) or HA^TMD-GFP (J) at the basal plane of cells, and DRAQ7 nuclear fluorescence intensity after 1 mM CuOOH treatment over time. The inhomogeneity distribution at each time point (D_t) was normalized to the distribution inhomogeneity at the initial time point of the experiment (D_i). Data information: All graphs show the mean ± SD. Data are representative of two (A, E) or three (F, G) independent experiments performed in triplicate, representative of 8 independent experiments (H), or pooled from two independent experiments performed in triplicate (B–D) or 8 (I) or 6 (J) independent experiments. Source data are available online for this figure.