Fig. 3: CaMKII activation and mPTP opening are essential for SSL10-induced necroptosis in HEK293T and HUVEC cells.

HEK293T or HUVEC cells were pretreated with 5 μM KN-93 for 30 min before SSL10 treatment, and release of LDH (a) and ATP (b) was determined. c CaMKII and phospho-Thr287 CaMKII were detected by immunoblotting in whole cell lysates of HEK293T cells treated with or without SSL10. d HEK293T or HUVEC cells were pretreated with 1 μM CsA for 30 min before SSL10 treatment, and the LDH release was detected. e Depolarization of the mitochondrial membrane of wild type (WT) HEK293T cells or Ripk3 knockout 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 are shown as a bar graph (right). Cells treated with 2 μM SSL7 were used as a negative protein control throughout the experiments. All data are presented as the means ± SD calculated 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 compared to the SSL10 treated group, by one-way (a–d) or two-way ANOVA (e).