Fig 2: Sur-X induced necroptosis by blocking the interaction of XIAP and TAB1 in colorectal cancer cells. a The crystal structure of TAB1-BIR1 complex obtained from PDB (code: 2POP) and colored by PyMol according to secondary structure. Helix in cyan, sheet in magenta and loop in wheat. b Analysis of interactions between XIAP and TAB1, TAB1 and TAK1, in HCT116 and RKO cells by co-immunoprecipitation. Three independent experiments were performed. c Effect of Sur-X on XIAP-TAB1 interaction as determined by co-immunoprecipitation. HCT116 cells were treated by 10 μM Sur-X for 1 h or not. Co-immunoprecipitation was performed by anti-XIAP antibody and anti-TAB1 antibody, respectively. XIAP and TAB1 were detected by Western blot analysis. GAPDH was used as a loading control. Anti-XIAP, anti-XIAP antibody; anti-TAB1, anti-TAB1 antibody. Three independent experiments were performed. d HCT116 and RKO cells were treated by 10 μM Sur-X (0.5, 1, 3 and 6 h) or Con (6 h), the expressions of TAB1, TAK1 and p-TAK1 were detected by Western blot analysis. GAPDH was used as a loading control. NT, no treatment. Three independent experiments were performed. e The protein expression of TAK1 in HCT116 cells with the treatment of 10 μM Sur-X or Con in combination with 10 μg/ml CHX for indicated time. GAPDH was used as a loading control. Three independent experiments were performed. f-g HCT116 was transiently transfected with NC and TAK1 siRNAs for 48 h, the expression of TAK1, p-RIP1 and p-MLKL were detected by Western blot analysis. GAPDH was used as a loading control. Three independent experiments were performed (f). HCT116 was transiently transfected with NC and TAK1 siRNAs for 48 h, followed by treatment of Sur-X for 6 h, and cell viability was detected by MTT assay, mean and SD of three independent experiments are shown. **, p < 0.01; ***, p < 0.001 (g). h-i Effect of TAB1 and TAK1 expression on the anticancer activity of Sur-X in HCT116 cells through necroptosis. HCT116 was transiently transfected with pcDNA3.1(+) and TAB1-OE plasmids for 24 h, followed by transfection of NC and TAK1 siRNA for 48 h. Transfected cells were treated by Sur-X for another 6 h and cell viability was detected by MTT assay, mean and SD of three independent experiments are shown. Comparison with NC: *, p < 0.05; ***, p < 0.001; ns, not significant. Comparison with TAB1-OE: #, p < 0.05; ##, p < 0.01; ####, p < 0.0001 (h). The expressions of TAB1, TAK1 and p-MLKL were detected by Western blot analysis. GAPDH was used as a loading control. TAB1-OE, TAB1-overexpression. Three independent experiments were performed (i). j Schematic representation of the anticancer mechanism of Sur-X

Fig 3: IDOAMP inhibits Aurora kinase A and promotes RIPK1/RIPK3/MLKL necrosome activation. (a) Inhibition of Aurora kinase A in the presence of IDOAMP after 24 h of incubation. (b) Co-IP analysis of the level of RIPK1 binding to FADD, RIPK3, and MLKL in PC3 cells. (c) Co-IP analysis of the levels of Aurora kinase A binding to RIPK1 and RIPK3 in PC3 cells following treatment with IDOAMP for 24 h. Data are presented as mean ± SD (n = 3). ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 vs. the control group.

Fig 4: HG-9-9-01 triggered RIPK1-RIPK3-Caspase 1-Caspase 8-GSDME dependent pyroptosis.a Relative viability of L929 cells treated with increasing concentrations of HG for indicated time points was determined by LDH. Bars represent the mean ± SEM from at least three independent experiments. b L929 cells were treated with indicated concentrations of HG for 6 h. Cell lysates were analyzed with immunoblotting. c, d L929 cells were transfected with specific siRNA targeting Gsdme and Gsdmd for 3 days and then treated with 5 μM HG for indicated time. Cell death was determined by released LDH. Knockdown efficiency was represented on the right side. c Bars represent the mean ± SEM from at least three independent experiments. d Cell lysates were used with immunoblotting analysis. e, f L929 cells were pretreated with indicated compounds for 30 min followed by treatment of 5 μM HG for 6 h. e Cell death was determined by released LDH. Bars represent the mean ± SEM from at least three independent experiments. f Cell lysates from (e) were performed with immunoblotting for cleaved Caspase-8 and GSDME. g L929 cells were transfected with indicated siRNA for 3 days. Cells were then stimulated with 5 μM HG for 6 h. Cell death was determined by released LDH, and the below panel showed the knockdown efficiency Bars represent the mean ± SEM from at least three independent experiments. h L929 cells were transfected with indicated siRNA for 3 days. Cells were stimulated with 5 μM HG for 6 h. Cell lysates were harvested and immunoblotted with indicated antibodies. i, j L929 cells were transfected with specific siRNA targeting caspase 1 and caspase 11 for 3 days and then treated with 5 μM HG for indicated time. Cell death was determined by released LDH, the below panel showed the knockdown efficiency. i Bars represent the mean ± SEM from at least three independent experiments. j Cell lysates were used with immunoblotting analysis.

Fig 5: Necroptosis is not the main pathway related to the death of ARPE-19 cells induced by SI.A Cell viabilities of ARPE-19 cells treated with 200 μM Nec-1, 2 μM NSA or 10 μM GSK′872 for 24 h before 20 mM NaIO3 treatment for 24 h (n = 4). B HT-29 cells with or without necrosulfonamide (NSA) pretreatment was treated with the indicated necrosis-inducing agents for 16 h. T + Z + S: TNF-α (200 ng/ml), Z-VAD (20 μM), and SM-164 (10 μM) (n = 4). C Phospho-RIPK1 (S166), total RIPK1, total RIPK3, Phospho-MLKL (S358), and total MLKL were detected in ARPE-19 or HT-29 cells by western blotting. SI: 10 mM NaIO3 treatment for 24 h. T + Z + S: TNF-α (100 ng/ml), Z-VAD (20 μM), and SM-164 (10 μM) treatment for 3 h. D Cell viabilities of SH-SY5Y cells treated with the indicated necrosis-inducer agent(s) for 3 h. T: 100 ng/ml TNF-α; S: 10 μM SM-164; Z: 20 μM Z-VAD (n = 4). E Cell viabilities of ARPE-19 cells treated with the indicated necrosis-inducer agent(s) for 48 h. T1: 50 ng/ml TNF-α; T2: 100 ng/ml TNF-α; S: 10 μM SM-164; Z: 20 μM Z-VAD (n = 4).