Figure 6.

MLKL-mediated calcium influx is involved in plasma membrane rupture during necroptosis. (a) HT29 cells were cultured in DMEM with or without calcium and treated with dimethylsulphoxide (DMSO) control, TSZ for 24 h or TS for 48 h. Cell survival was determined by PI staining. Results shown are averages ± s.e.m. from three independent experiments. (b) HT29 cells were loaded with Fluo4 AM and then treated with TSZ in the presence of normal medium or calcium-free medium for 4 h. Representative confocal images of live HT29 cells are shown. (c) HT29 shRNA control cells or MLKL shRNA cells were loaded with Fluo4 AM and then treated with TSZ for 4 h. Representative confocal images of live HT29 cells are shown. (d) Left, HT29 MLKL-shRNA cells were transfected with DsRed–MLKL-WT, DsRed–MLKL-CC1, DsRed–MLKL-CC2 or DsRed–MLKL-T357A/S358A as indicated. After 24 h transfection, cells were loaded with Fluo4 and then treated with TSZ for 4 h. The representative confocal images are shown. Right, ratiometric measurement of Fluo4 mean fluorescence intensity between transfected and non-transfected cells (20 cells from each part). Results shown are averages ± s.e.m. from three independent experiments. (e) MEF cells were cultured with or without calcium or pre-treated with BAPTA-AM (10 μm) for 30 min and then treated with TCZ for 13 h. Cell survival was determined by PI staining. Results shown are averages ± s.e.m. from three independent experiments. (f) WT, MLKL-deficient or RIP3-deficient MEF cells were treated with TSZ or TCZ for 6 h. Cells were collected and loaded with Fluo4. Fluo4 fluorescent cells were determined by FACS analysis. **P < 0.01. Scale bar, 10 μm. Statistics source data for this figure can be found in Supplementary Table 1.