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. 2024 Feb 9;43(6):904–930. doi: 10.1038/s44318-024-00044-1

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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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Figure EV2 — (A) SVEC4-10 cells treated for 3 h with 10 μΜ ABT-737, 10 μΜ S63845 and 30 μΜ Q-VD-OPh. Mitochondria were isolated using Dounce homogeniser and cellular fractions were probed with relevant antibodies. Mitochondrial localised NEMO was quantified normalising to mitochondrial content defined by HSP60 signal. (B) SVEC4-10 cells pre-treated with 2 μΜ TAK-243 for 1 h followed by additional 1 h treatment with 10 μΜ ABT-737, 10 μΜ S63845 and 30 μΜ Q-VD-OPh with or without 2 μΜ TAK-243. Blots are representative for four independent experiments. (C) Upper: SVEC4-10 cells expressing GFP-NEMO were pre-treated for 1 h with 2 μΜ TAK-243 followed by 1 h treatment of 10 μΜ ABT-737, 10 μΜ S63845, 30 μΜ Q-VD-OPh with or without 2 μΜ TAK-243. Cells were immunostained for TOM20 and ubiquitin (FK2). Scale bar is 50 μm and images are representative for three independent experiments. Lower: quantification showing the percentage of cells with mitochondrial localised GFP-NEMO and ubiquitin puncta. (D) SVEC4-10 cells pre-treated with 1 μΜ MLN4924 (NAE inhibitor) for 1 h followed by additional 1 h treatment with 10 μΜ ABT-737, 10 μΜ S63845 and 30 μΜ Q-VD-OPh with or without 1 μΜ MLN4924. Blots are representative for two independent experiments. (E) SVEC4-10 cells expressing GFP-NEMO pre-treated with 1 μΜ MLN4924 for 1 h followed by additional 1 h treatment with 10 μΜ ABT-737, 10 μΜ S63845 and 30 μΜ Q-VD-OPh with or without 1 μΜ MLN4924. Cells were immunostained for mitochondrial TOM20 and DAPI. Images are representative for three independent experiments and are shown with a 50 μm scale bar. (F) Quantification of (E) showing the percentage of cells with mitochondrial translocation of GFP-NEMO. Data information: graphs in (C, F) display mean values ± s.e.m. (error bars) of n = 3 independent experiments. Statistics were performed using two-way ANOVA with Tukey correction. ****P < 0.0001.