Figure 1HT-29 cells were transfected with siRNA for MLKL or scramble non-specific (NS) siRNA for 72 hours. Cells pre-treated with 10 μM QVD-OPh (Q), 5 μM Birinapant (S), 20 μM necrostatin-1 (Nec-1s) were stimulated with 1-10 ng.mL-1 TNFα (T). Cell viability was assessed by CellTiter-Glo (A) after 16 hours. Data are means ± SEM of three independent experiments. *P< 0.1, ****P<0.0001 (ANOVA).HT-29 cells were transfected with siRNA for MLKL or scramble non-specific (NS) siRNA for 72 hours. Cells pre-treated with 10 μM QVD-OPh (Q), 5 μM Birinapant (S), 20 μM necrostatin-1 (Nec-1s) were stimulated with 1-10 ng.mL-1 TNFα (T). Cell viability was assessed by Crystal Violet (B) after 16 hours. Data are means ± SEM of three independent experiments. *P< 0.1, ****P<0.0001 (ANOVA).HT-29 cells were transfected with siRNA for MLKL or scramble non-specific (NS) siRNA for 72 hours. Cells pre-treated with 10 μM QVD-OPh (Q), 5 μM Birinapant (S), 20 μM necrostatin-1 (Nec-1s) were stimulated with 1-10 ng.mL-1 TNFα (T). Flow cytometric analysis of cells stained with TO-PRO-3 nd Annexin V (A5). Histograms show means ± SEM of three independent experiments. ***P<0.001, ****P<0.0001 (ANOVA).HT-29 cells were transfected with siRNA for MLKL or scramble non-specific (NS) siRNA for 72 hours. Cells pre-treated with 10 μM QVD-OPh (Q), 5 μM Birinapant (S), 20 μM necrostatin-1 (Nec-1s) were stimulated with 1-10 ng.mL-1 TNFα (T). Flow cytometric analysis of cells stained with Annexin V (A5). Histograms show means ± SEM of three independent experiments. ***P<0.001, ****P<0.0001 (ANOVA).HT-29 cells were transfected with siRNA for MLKL or scramble non-specific (NS) siRNA for 72 hours. Cells pre-treated with 10 μM QVD-OPh (Q), 5 μM Birinapant (S), 20 μM necrostatin-1 (Nec-1s) were stimulated with 1-10 ng.mL-1 TNFα (T). Flow cytometric analysis of cells stained with TO-PRO-3 Histograms show means ± SEM of three independent experiments. ***P<0.001, ****P<0.0001 (ANOVA).HT-29 cells were transfected with siRNA for MLKL or scramble non-specific (NS) siRNA for 72 hours. Cells pre-treated with 10 μM QVD-OPh (Q), 5 μM Birinapant (S), 20 μM necrostatin-1 (Nec-1s) were stimulated with 1-10 ng.mL-1 TNFα (T). Cells were collected after 4 and 24 hours of treatment, and stained with TO-PRO-3 Data are means ± SEM of three independent experiments. **P<0.01, ***P< 0.001, ****P<0.0001 (ANOVA).HT-29 cells were transfected with siRNA for MLKL or scramble non-specific (NS) siRNA for 72 hours. Cells pre-treated with 10 μM QVD-OPh (Q), 5 μM Birinapant (S), 20 μM necrostatin-1 (Nec-1s) were stimulated with 1-10 ng.mL-1 TNFα (T). Cells were collected after 4 and 24 hours of treatment, and stained with TO-PRO-3 Data are means ± SEM of three independent experiments. **P<0.01, ***P< 0.001, ****P<0.0001 (ANOVA).HT-29 cells were transfected with siRNA for MLKL or scramble non-specific (NS) siRNA for 72 hours. Cells pre-treated with 10 μM QVD-OPh (Q), 5 μM Birinapant (S), 20 μM necrostatin-1 (Nec-1s) were stimulated with 1-10 ng.mL-1 TNFα (T). Cells were collected after 4 hours of treatment, and stained with propidium iodide (PI) and A5 Data are means ± SEM of three independent experiments. **P<0.01, ***P< 0.001, ****P<0.0001 (ANOVA).HT-29 cells were transfected with siRNA for MLKL or scramble non-specific (NS) siRNA for 72 hours. Cells pre-treated with 10 μM QVD-OPh (Q), 5 μM Birinapant (S), 20 μM necrostatin-1 (Nec-1s) were stimulated with 1-10 ng.mL-1 TNFα (T). Cells were collected after 4 and 24 hours of treatment, and stained with propidium iodide (PI) Data are means ± SEM of three independent experiments. **P<0.01, ***P< 0.001, ****P<0.0001 (ANOVA).HT-29 cells were transfected with siRNA for MLKL or scramble non-specific (NS) siRNA for 72 hours. Cells pre-treated with 10 μM QVD-OPh (Q), 5 μM Birinapant (S), 20 μM necrostatin-1 (Nec-1s) were stimulated with 1-10 ng.mL-1 TNFα (T). J Measurement of lactate deshydrogenase (LDH) released from necroptotic cells, as indicated (means ± SEM, n=4 biological replicates, ****P<0.0001, ANOVA).HT-29 cells were transfected with siRNA for MLKL or scramble non-specific (NS) siRNA for 72 hours. Cells pre-treated with 10 μM QVD-OPh (Q), 5 μM Birinapant (S), 20 μM necrostatin-1 (Nec-1s) were stimulated with 1-10 ng.mL-1 TNFα (T). K Time lapsed microscopy analysis of A5 and TO-PRO-3 staining upon induction of necroptosis. The presented data are representative of at least three independent experiments.