MLKL Oligomerization and Vesicular Trafficking enable Plasma Membrane "Leakiness".Figure 3HT-29 cells were transfected with a nonspecific (NS) siRNA or with the indicated siRNAs for 72 hours. Cells were pre-treated with 10 μM QVD-OPh (Q) plus 5 μM Birinapant (S), and exposed to 10 ng.mL-1 of TNFα (T). NS- and MLKL-silenced cells were treated with 5 μM necrosulfonamide (NSA). MLKL oligomers (MLKLn) were visualized by non-reducing SDS-PAGE after cross-linking proteins (A). Data are means ± SEM of three independent experiments. ****P<0.0001 (ANOVA). Data information Arrowhead, MLKL cleaved fragment. HT-29 cells were transfected with a nonspecific (NS) siRNA or with the indicated siRNAs for 72 hours. Cells were pre-treated with 10 μM QVD-OPh (Q) plus 5 μM Birinapant (S), and exposed to 10 ng.mL-1 of TNFα (T). NS- and MLKL-silenced cells were treated with 5 μM necrosulfonamide (NSA). MLKL oligomers (MLKLn) Cells were exposed to TQS for 5 hours. Molecular weight markers (Mr) are shown. TO-PRO-3 uptake was analyzed by flow cytometry in cells treated with TQS for 4 hours (B). Data are means ± SEM of three independent experiments. ****P<0.0001 (ANOVA). HT-29 cells were transfected with a nonspecific (NS) siRNA or with the indicated siRNAs for 72 hours. Cells were pre-treated with 10 μM QVD-OPh (Q) plus 5 μM Birinapant (S), and exposed to 10 ng.mL-1 of TNFα (T). C Cell lysates from NS-, and ITPK1-silenced cells were analyzed by Western blotting as indicated. Data information: Arrowhead, MLKL cleaved fragment. HT-29 cells were transfected with a nonspecific (NS) siRNA or with the indicated siRNAs for 72 hours. Cells were pre-treated with 10 μM QVD-OPh (Q) plus 5 μM Birinapant (S), and exposed to 10 ng.mL-1 of TNFα (T). D Western blotting analysis of MLKL oligomerization HT-29 cells were transfected with a nonspecific (NS) siRNA or with the indicated siRNAs for 72 hours. Cells were pre-treated with 10 μM QVD-OPh (Q) plus 5 μM Birinapant (S), and exposed to 10 ng.mL-1 of TNFα (T). E Cell survival was evaluated by CellTiter-Glo after 24 hours of treatment (means ± SEM, n=4 biological replicates, ****P<0.0001, ANOVA). HT-29 cells were transfected with a nonspecific (NS) siRNA or with the indicated siRNAs for 72 hours. Cells were pre-treated with 10 μM QVD-OPh (Q) plus 5 μM Birinapant (S), and exposed to 10 ng.mL-1 of TNFα (T). F Measurement of TO-PRO-3 uptake as in (B). Dead TO-PRO-3high cells were discarded. Data are means ± SEM of three independent experiments. *P<0.1, ****P<0.0001 (ANOVA). HT-29 cells were transfected with a nonspecific (NS) siRNA or with the indicated siRNAs for 72 hours. Cells were pre-treated with 10 μM QVD-OPh (Q) plus 5 μM Birinapant (S), and exposed to 10 ng.mL-1 of TNFα (T). G Small EVs fractions were purified from the culture medium by ultracentrifugation. Shown is a representative diagram of particle count obtained with tunable resistive pulse sensing analysis (TRPS, qNano, IZON). HT-29 cells were transfected with a nonspecific (NS) siRNA or with the indicated siRNAs for 72 hours. Cells were pre-treated with 10 μM QVD-OPh (Q) plus 5 μM Birinapant (S), and exposed to 10 ng.mL-1 of TNFα (T). H Cells were subjected to Western blotting analysis Data information: Arrowhead, MLKL cleaved fragment. HT-29 cells were transfected with a nonspecific (NS) siRNA or with the indicated siRNAs for 72 hours. Cells were pre-treated with 10 μM QVD-OPh (Q) plus 5 μM Birinapant (S), and exposed to 10 ng.mL-1 of TNFα (T). I MLKL oligomerization was determined Data information Arrowhead, MLKL cleaved fragment. HT-29 cells were transfected with a nonspecific (NS) siRNA or with the indicated siRNAs for 72 hours. Cells were pre-treated with 10 μM QVD-OPh (Q) plus 5 μM Birinapant (S), and exposed to 10 ng.mL-1 of TNFα (T). J Cell survival by CellTiter-Glo after 24 hours (mean ± SEM, n=4 biological replicates, **P<0.01, ***P<0.001, ****P<0.0001, ANOVA). HT-29 cells were transfected with a nonspecific (NS) siRNA or with the indicated siRNAs for 72 hours. Cells were pre-treated with 10 μM QVD-OPh (Q) plus 5 μM Birinapant (S), and exposed to 10 ng.mL-1 of TNFα (T). K TO-PRO-3 uptake Data are means ± SEM, n=3 biological replicates, ****P<0.0001 (ANOVA). The presented data are representative of at least three independent experiments.