Fig 2: Smac mimetics reduce adverse toxicity of MTAs by potentiating memTNF-mediated apoptosis. a Immunoblotting analysis of apoptosis markers using whole cell lysates from MTA-treated HeLa cells. Cells were treated with 1 µM NCZ or PTX for indicated time. b Immunoblotting analysis of apoptosis markers using whole cell lysates from MTAs and LCL161 co-treated HeLa cells. Cells were treated with 100 nM MTAs or 20 ng/ml recombinant/soluble TNF (T) in the presence or absence of 100 nM LCL161 as indicated for 20 h. c Time course effect of LCL161 on MTA-induced cell death in HeLa cells. d Effect of TNFR1 knockout on MTAs and LCL161 co-treatment induced apoptosis in HeLa cells. Cells were treated as indicated for 28 h. e Effect of TNF neutralization on MTAs and LCL161 co-treatment induced cell death in HeLa cells. E, Enbrel. f Dose-dependent effect of NCZ (upper) or PTX (lower) treatment on HeLa cells in the presence or absence of 100 nM LCL161 (left), 100 nM GDC-0917 (middle), or 100 nM GDC-0152 (right). D, DMSO; NCZ, nocodazole; PTX, paclitaxel; LCL, LCL161; Z, z-VAD. Cell viability was determined by measuring ATP levels. The data are represented as mean ± SEM of duplicate wells (a, c–e). Results are reported from one representative experiment. Experiments were repeated independently three (b–e, and LCL161 in f) or two (a, GDC-0917 and GDC-0152 in f) times

Fig 3: APG-1387 induces cell death partly through TNF-alpha signaling pathway. a ELISA analysis of the secretion of TNF-alpha after different time points of APG-1387 on SKOV3 cells streatment. b Cells were treated with different time points of APG-1387, and the secretion of TNF-alpha were analyzed by ELISA. c Cells were transfected with TNFR1 siRNAs, cell viability was determined by the CCK-8 assay. d Cells were transfected with TNF-alpha siRNAs, Cell viability was determined by the CCK-8 assay. e APG-1387 was coadministered with or without TNF-alpha neutralizing antibody, cell viability was determined by the CCK-8 assay. f Cells were treated with different concentrations of APG-1387 on cells after 24 h treatment and the expression levels of NF-?B1/p50 and NF-?B2/p52 were determined by western blot. g The protein levels of NF-?B1/p50 and NF-?B2/p52 in cells by western blot after APG-1387 was coadministered with or without Necrostatin-1

Fig 4: Model. TNF-R1 is palmitoylated in the ER (PATx) or Golgi (PATy) and transported to caveolae in the plasma membrane. After TNF binding, TNF-R1 is either de-palmitoylated by APT2, resulting in translocation to another lipid raft compartment, complex I formation and signaling for NF-?B activation. Alternatively, TNF-R1 may translocate out of caveolae and TNF-receptosomes are formed by clathrin dependent internalization. This allows complex II/DISC formation by intracellular maturation. In case of APT2 depletion/inhibition, activated TNF-R1 induces nSMase dependent ceramide production in caveolae which triggers cell death

Fig 5: Schematic illustrations of how necroptosis and apoptosis were regulated in NEC.In NEC patients or cells challenged by LPS, TLR4 is activated. On the one hand, TLR4 binds to TRIF to activate necroptosis. On the other hand, TLR4 activation induces the production of inflammatory cytokines including TNF-a, which further binds with TNFR1 and leads to apoptosis and necroptosis simultaneously. When RIPK1 is inhibited by necrostatin-1s, or when TLR4 is removed, necroptosis becomes suppressed. The production of TNF-a is also reduced so that caspase-8 is decreased, leading to a decreasing trend of apoptosis. Meanwhile, the suppression of RIPK1 might relieve its inhibitory effect on caspase-3, leading to an increased trend of apoptosis. These two effects may counteract each other, but in general necrostatin-1 and TLR4 knockout showed an alleviating effect on NEC mice.