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. 2023 Nov 13;30(11):1806–1815. doi: 10.1038/s41594-023-01136-y

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© The Author(s) 2023

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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Extended Data Fig. 3 — a. Immunoblot analysis of FSP1 (using AMID (Santa Cruz) and HA antibodies) and VCP expression in Pfa1 cells stably overexpressing wildtype hFSP1 or its mutant variants. For immunoblot of FSP1, s.e. and l.e. represent short and long exposure, respectively. b. Crystal structure of Saccharomyces cerevisiae (S. cerevisiae) NDH-2 (PDB: 4G73) with its cofactors, FAD (yellow), NADH (blue), and CoQ₅ (green). The proton transfer via sequential carboxylic residues in the α-helix (that is, E242/E246/D249/D294) and subsequent final protonation from Y482 to ubiquinone are indicated with black arrows. c. Crystal structure of Caldalkalibacillus thermarum (C. thermarum) NDH-2 (PDB: 4NWZ) with its cofactors, FAD (yellow), NADH (blue), and CoQ₅ (green). The proton transfer via sequential carboxylic residues in the α-helix (that is, E169/E173/D176/E180) and subsequent final protonation from K376 to ubiquinone are indicated with black arrows. d. Cell viability was measured after treating Pfa1 cells stably overexpressing wildtype hFSP1 or its mutant variants with or 300 nM RSL3 for 24 h. Data was normalized by each group of non-treatment with RSL3. Data represents the mean ± SD of 3 wells of a 96-well plate from one out of 3 independent experiments. e. Immunoblot analysis of FSP1 (Santa Cruz) and VCP expression in Pfa1 cells stably overexpressing wildtype hFSP1 or its mutant variants in cells. Data is shown from a single experiment (a,e).

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