Fig. 8

TNF mediated activation of necroptosis in human iPSC-derived glutamatergic neurons when apoptosis is inhibited. a Viability of human iPSC-derived glutamatergic neurons treated with combinations of TNF (100 ng/mL), SMAC mimetic (2 µM), and zVAD (10 µM) [TSZ], or vehicle control, assessed by LDH assay at 24 h. b Viability of human iPSC-derived glutamatergic neurons treated with vehicle control, TSZ, or TSZ with GSK-547 (10 nM), GSK-872 (100 nM), or necrosulfonamide (NSA, 1 µM), assessed by LDH assay at 24 h. c Quantification of intracellular ROS in human iPSC-derived glutamatergic neurons treated with vehicle control, TSZ, or TSZ with GSK-547, GSK-872, or NSA. d Quantification of neurite beading in human iPSC-derived glutamatergic neurons treated with vehicle control, TSZ, or TSZ with GSK-547, GSK-872, or NSA. e Representative images of human iPSC-derived glutamatergic neurons treated with vehicle or TSZ immunostained for pMLKL (red) and DAPI (blue), along with representative images of TSZ treated neurons co-stained for TNFR1 (green) and pMLKL (pink), RIPK1 (white) and pMLKL (pink), and pRIPK3 (yellow) and pMLKL (pink) (scale bar = 20 µm). f Quantification of pMLKL + neurons treated with vehicle control, TSZ, or TSZ with GSK-547, GSK-872, or NSA. g Quantification of Chmp2b mean intensity normalized with DAPI mean intensity. All quantification performed with 3 replicates per group per experiment. One-way ANOVA with Bonferroni’s post-hoc correction for multiple group comparisons, and t-test for comparing between two groups. Data are represented as mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001