Fig. 5: Necroptosis is accelerated between junctioned epithelial cells.

a HT29 cells were TNF, Smac mimetic and IDN-6556 (TSI)-treated, then Annexin V-binding (green) and SYTOX Green-uptake (red) were imaged via time-lapse epifluorescence microscopy. b, c HT29 cells were treated with TSI (necroptotic stimulus) or TS (apoptotic stimulus), then propidium iodide-uptake (death) and subsequent movement of dead cells was tracked over time using the IncuCyte S3 System. d Each dot represents the spatiotemporal gap between two cell death events. Mean ± 95% confidence intervals; dashed line represents the average spatiotemporal gap between all cell death events across the population; ^n.s.p = 0.2223, *p = 0.0422 and ***p = 0.0006 via one-way ANOVA with Tukey’s correction for multiple comparisons. For TSI-treated HT29 cells: N = 188, 289, 298 gaps for the respective 1, 2, 3-cell-width groups and N = 22676 gaps for the whole population. For TS-treated HT29 cells: N = 110 gaps for 1-cell-width group and N = 17838 gaps for the whole population. e Co-cultures of TSI-treated wild-type (expressing mTagRFP-Membrane-1 fusion protein) and MLKL^−/− (diffuse expression of mCherry) HT29 cells were imaged via time-lapse lattice light sheet microscopy (LLSM) with staining for Annexin V-binding (top row; yellow signal) and markers (bottom row; same as top row but with inverted signal). MIP micrographs are representative of N = 7 MLKL^−/− cells across n = 3 independent experiments. TSI-treatment times are shown; magenta lines demarcate MLKL^−/− cell boundaries.