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. 2020 Jun 19;11:3151. doi: 10.1038/s41467-020-16887-1

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© The Author(s) 2020

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 — a Images of TSI-treated HT29 cells expressing Mb37 stained for MLKL^10C2, MLKL^pS358 and DNA (Hoechst). b Mean frequency of clusters and hotspots from n = 2 independent experiments (N > 1500 cells per group) with raw lactate dehydrogenase (LDH) release values taken from the same samples. c–g Necrosulfonamide (NSA) was added to HT29 cells together with TNF, Smac mimetic and IDN-6556 (TSI) or 4–6 h after TSI addition. Plasma membrane lysis (LDH release; line shows mean of 4 biological replicates indicated as dots; data representative of n = 3 independent experiments) (d), MLKL activation (e) and hotspots (f, g) were measured after 7.5 h of TSI-treatment. e Cytosol (C) and membrane (M) fractions resolved by Blue Native-PAGE and immunoblotted for MLKL and MLKL^pS358. Uncropped blots are included as Source Data. Fractionation was verified by probing for voltage-dependent anion channel 1 (VDAC1; membrane), glyceraldehyde-3-phosphosphate dehydrogenase GAPDH (cytosol); representative of n = 2 independent experiments. f Mean frequency, intensity and size of hotspots from one experiment (N = 1309-1740 cells per group); representative of n = 3 independent experiments.