Fig. 4

Sur-X promoted necroptosis in colorectal cancer cells. a Cell morphology as determined by Giemsa staining. After treated by 10 μM Sur-X or Con, HCT116 cells were stained with Giemsa and those treated by adriamycin (10 μM for 12 h) was used as a positive control of apoptosis. Scale bar, 50 μm. Three independent experiments were performed. b The real-time detection of RFU (cell membrane damage, necrosis) in HCT116 cells over 6 h with indicated concentrations of Sur-X. NT, no treatment. Three independent experiments were performed. c HCT116 (top) and RKO (bottom) cells were treated by 10 μM Sur-X for 1, 3 and 6 h, or Con for 6 h, and analyzed by Annexin V/7-AAD assay (left panel). Quantification of 7-AAD positive cells, mean and SD of three independent experiments are shown (right panel). 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 necroptosis-related proteins were detected by Western blot analysis. GAPDH was used as a loading control. NT, no treatment. Three independent experiments were performed. e-f Effect of Nec-1 s-pretreatment on Sur-X-induced necroptosis in HCT116 (top) and RKO (bottom) assessed by Annexin V/7-AAD assay (e). Quantification of Annexin V positive cells and quantification of 7-AAD positive cells in HCT116 (top) and RKO (bottom), mean and SD of three independent experiments are shown. NT, no treatment; Nec-1 s, cells were treated only by Nec-1 s; Sur-X, cells were treated by Sur-X (10 μM) for 6 h; Nec-1 s + Sur-X, cells were pretreated by Nec-1 s (50 μM) for 12 h and treated by Sur-X in combination with Nec-1 s for another 6 h (f). g Kinetic detection of apoptosis (RLU, phosphatidylserine and Annexin V binding) and necroptosis (RFU, membrane integrity) in HCT116 cells treated by 10 μM Sur-X was conducted simultaneously over 6 h. Three independent experiments were performed. **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; ns, not significant