Fig. 5

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