Fig. 2: Death coincides with necrosomal and plasma membrane hotspot MLKL accumulation.
a Homology model of full-length human MLKL66 highlighting the 7G2, 10C2 and pS358 antibody and Monobody 37 (Mb37)37 epitopes, and the target of necrosulfonamide (NSA), C866. b Model for immunofluorescent detection of the stages of RIPK1 and MLKL activation. c–d TSI-treated HT29 cells stained for MLKL7G2, MLKLpS358 and DNA (Hoechst). Imaged by epifluorescence and differential interference contrast (DIC) microscopy. Representative of n = 4 independent experiments. c MLKL7G2 clusters, MLKLpS358 hotspots and cell lysis emerge >4.5 h post-TSI treatment. d Mean frequency, intensity and size of clusters and hotspots during TSI-induced necroptosis from one experiment (N = 1258-1973 cells per timepoint). e 3D-SIM maximum intensity projection (MIP), Z-section and zoomed micrographs of TSI-treated HT29 cells revealed MLKLpS358 puncta (white arrow) located basally to a MLKLpS358 hotspot. The hotspot colocalizes with the Wheat germ agglutinin (WGA)-stained plasma membrane of an intercellular junction (yellow arrow) apical to MLKL10C2 clusters (magenta arrow). f En face view of three different MLKLpS358 hotspots of heterogeneous substructure. g TSI-treated HT29 cells were stained for MLKL10C2, RIPK1, ZO-1 (a proxy for the MLKLpS358 immunosignal; refer to Fig. 8a), and DNA (Hoechst), and imaged by 3D Airyscan microscopy (representative of n = 3 independent experiments).
