Fig. 5

Necrotic NK cell cytotoxicity is triggered by granzyme and mediated by the necroptosis pathway. a Representative phase-contrast and the corresponding GrzmB-FRET reporter images of MCF7 cells in co-culture with primary NK cells with or without 10 nM CMA (vacuolar-type ATPase inhibitor that disrupts lytic granules). Time is indicated in the unit of hours:minutes on the GrzmB-FRET reporter images. The white scale bar is 20 μm. b Distribution of the indicated response phenotypes of MCF7 cells after 6-h co-culture with primary NK cells under the control condition or treatment of 10 nM CMA, 0.9 mM EGTA (calcium chelator that inhibits lytic granule transfer), 40 μM DCI (pan-granzyme inhibitor), and 50 μM Ac-IEPD-CHO (granzyme B inhibitor). c Distribution of the cytotoxic response phenotypes of MCF7 cells upon knockdown of RIP1, RIP3, and MLKL, respectively, and under the treatment of 5 μM GSK’872 (RIP3 inhibitor). The knockdown efficiency is demonstrated by the western blots. Data plotted in b and c were averaged from 3 independent imaging experiments, and the number of cells analyzed for each condition/experiment ranges from 55 to 112. The error bars are standard deviations. Data acquired with NK cells from the same donors are denoted with the same color symbols in each sub-figure. P values were obtained by Student’s t test comparing the treatment condition with control. *P < 0.001. d Left panel: western blot analysis of phospho-MLKL upregulation in MCF7 cells in co-culture with primary NK cells for 3 h and 6 h, respectively. Right panel: representative images of phospho-MLKL immunofluorescence signal in MCF7 cells under control vs. in co-culture with primary NK cells. The white scale bar is 5 μm