Figure 4.

MLKL translocation as a downstream event of RIP3 was required in matrine-induced necroptosis. (a and b) MLKL was required in matrine-induced necroptosis in Mz-ChA-1 (a) and QBC939 (b) cells. Cells were pre-treated with MLKL inhibitor NSA (20 nM) for 2 h, and then treated with matrine (1.5 mg/ml) or vehicle for 48 h. After that, the percentage of cell death was determined by PI staining and flow cytometry. Results were presented as the mean±S.D. from three independent experiments. Significant differences were indicated as *P<0.05, **P<0.01 and ***P<0.001 (assessed by Student’s t-test). (c) MLKL translocation from cytoplasm to plasma membrane induced by matrine were blocked by Nec-1. Mz-ChA-1 and QBC939 cells were pre-treated with Nec-1 (20 μM) for 2 h, and then treated with matrine (1.5 mg/ml) or vehicle for another 2 h. MLKL subcellular localization was analyzed by immunofluorescence and confocal laser scanning microscopy.