Fig. 4: MLKL tetramerization is essential for necroptotic cell death.

A, B Liposome dye release assays on recombinant human MLKL protein complexes. A Tetrameric MLKL (unliganded, stabilized by Mb27, or phosphorylated by RIPK3) are considerably more effective at permeabilizing liposomes than monomeric MLKL (apo, or TSEE mutant in 1:1 complex with RIPK3). B The brace helix suppresses the membrane permeabilization activity of 4HB domain in monomeric MLKL. C Evaluation of necroptotic signalling by wild-type and tetramerization interface mutants of full-length human MLKL in MLKL^-/- HT29 cells. Human MLKL expression (wild-type or mutant) was induced with doxycycline (Dox; 100 ng/mL) and cell death was measured in the presence or absence of necroptotic stimulus (TNF, Smac mimetic, IDN-6556; TSI) at 24 h. Cell death was quantified as a percentage of SYTOX Green positive cells using IncuCyte SX5 live cell imaging. Two independent cell lines were generated for wild-type and MLKL mutants. One independent line was assayed in n = 1 and the other in n = 2, for a total of n = 3 independent experiments. Data are plotted as mean ± SEM. D Cytosol (c) and membrane (m) fractions resolved by Blue Native-PAGE from transduced MLKL^-/- HT29 cells treated with doxycycline (Dox, 100 ng/mL) in the presence or absence of necroptotic stimulus (TSI; 4.5 h). Fractionation was verified by probing for VDAC (membrane) and GAPDH (cytosol) via SDS-PAGE. Images are representative of n = 2 independent experiments. E MLKL^-/- HT29 cells stably transduced with MLKL^WT or MLKL^R30E were treated with doxycycline (Dox) only or doxycycline plus necroptotic stimulus (Dox, TSI) in the presence or absence of MLKL inhibitor necrosulfonamide (NSA) or RIPK3 kinase inhibitor GSK872. IncuCyte SX5 imaging quantified the percentage of cell death (SYTOX Green-positive cells) every hour for 23 h. Data are plotted as mean ± SEM for n = 4 for wild-type MLKL and n = 6 for R30E MLKL. Two independent cell lines were generated for each of the wild-type and MLKL R30E mutant; WT cell lines were assayed in n = 2 and R30E mutant cell lines were assayed in n = 3 independent experiments.