Fig. 5: Acrolein hinders necroptosis via targeting MLKL oligomerization.

A, B Immunoblot analysis of mixed lineage kinase domain-like pseudokinase (MLKL) oligomers by detecting total MLKL (tMLKL) in non-reducing condition and immunoblot analysis of phosphorylated MLKL (pMLKL) and tMLKL in bone marrow-derived macrophages (BMDMs) after indicated treatments. The single asterisk (*) indicates the band for potential higher molecular weight aggregates and the double asterisks (**) indicate the band for reduced monomer of MLKL. TSZ [murine TNF, Smac-mimetic and Z-VAD-FMK (zVAD)], necrostatin-1 (Nec-1), dithiothreitol (DTT), acrolein (Acr), and ferrostatin-1 (Fer-1) were used. β-actin was used as an internal control. C Real-time analysis of cell death of BMDMs with TSZ as described in the presence of GSK’872 (GSK) or Acr combined with Fer-1. D Immunoblot analysis of MLKL oligomers by detecting total MLKL under non-reducing conditions and immunoblot analysis of pMLKL and tMLKL in TSZ (human TNF, Smac-mimetic, and zVAD)-treated HT-29 cells with or without Nec-1 and Acr. E, F Real-time analysis of cell death in TSZ-treated (E) HT-29 cells and (F) CT-26 cells with or without Acr and Nec-1. G Representative images of MLKL immunofluorescence in TSZ-treated L929 cells and HT-29 cells as described in the presence or absence of Acr. White arrowheads indicate MLKL puncta. H Real-time analysis of cell death in RIPK3-expressing HeLa cells exposed to N¹,N¹¹-diethylnorspermine (DENSPM), genetically modified using CRISPR-Cas9 with either a non-targeting scramble guide RNA (Scr) or SAT1-specific guide RNAs (SAT1^−/− #g1 and SAT1^−/− #g2), treated with TSZ (human TNF, Smac-mimetic BV-6 and zVAD) in the presence or absence of Nec-1. Nec-1, GSK, Fer-1, and Acr were pretreated before TSZ stimulation. BV-6 was used in experiments where specifically noted. Data are representative of at least three independent experiments (A, B, D, and G). Data are shown as mean ± SEM; ****P < 0.0001 (two-way ANOVA; n = 4 from 4 biologically independent samples) (C, E, F, and H). Scale bar, 20 μm (G). Source data are provided as a Source Data file.