Fig. 4: SSL10 induces necroptosis by direct interacting with the extracellular domain of TNFR1 (TNFR1ECD).
a Wild type (WT) or Tnfrsf1a knockout (KO) HEK293T cells were treated with SSL10 at different concentrations for 30 min, and the SSL proteins bound to the cell surface were detected by flow cytometry after FITC-conjugated anti-His-tag antibody staining. b WT or Tnfrsf1a KO cells were treated with 2 μM SSL10 for 48 h at 37 °C and 5% CO2, and the LDH release was detected. c WT or Tnfrsf1a KO HEK293T cells were challenged with 2 μM SSL10 for 48 h and the cell viability was measured by flow cytometry after Annexin V/PI staining. The dot plot (left) is representative of three independent experiments, and the results of quantification (right) are shown as a bar graph. d Depolarization of the mitochondrial membrane of WT or Tnfrsf1a KO HEK293T cells treated with SSL10 was measured by flow cytometry after JC-1 staining. The dot plot (left) is representative of three independent experiments, and the results of quantification (right) are shown as a bar graph. e Immunoblotting of SSL10 after pull-down with MBP-TNFR1ECD. f ITC assays for SSL10 binding to MBP-TNFR1ECD or MBP control protein. Cells treated with 2 μM SSL7 were used as a negative protein control throughout the experiments. All data are presented as the means ± SD from three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001 compared to the Ctrl cells (buffer-treated cells). #p < 0.05; ##p < 0.01; ###p < 0.001 compared to the SSL10 treated group, by two-way ANOVA.
