Fig. 3

Seasonal NC/99 IAV induces RIPK3-dependent apoptosis and necroptosis. a DC infected for 8 h with either mock or NC/99 IAV were pretreated for 1 h with various pharmacological inhibitors: Z-VAD-FMK (20 μM), necrosulfonamide (NSA; 15 μM), GSK872 (5 μM), GSK840 (5 μM), and necrostatin (Nec-1; 30 μM). Percentage of cell death was determined by imaging flow cytometry. b Genetic ablation of RIPK1, RIPK3, MLKL, FADD, and DAI, either alone or in combination, was carried out by electroporation of DC with CRISPR/Cas9 ribonucleoproteins. Electroporated DC (negative control), DC electroporated with Cas9 Nuclease in the absence of targeting guide RNAs. Following an 8-h infection with either mock or NC/99 IAV, cell death was assayed in DC selected for having low levels of expression of the ablated gene(s) by imaging flow cytometry analysis (Supplementary Fig. 3). c, d Imaging flow cytometry measurements of MLKL phosphorylation and caspase 8 cleavage following a 5-h infection with either mock, NC/99, or Cal/09 IAV at the indicated multiplicities of infection (MOIs). DC treated for 5 h with the combination of TNFα (0.1 μg/ml), cycloheximide (2 μg/ml), and Z-VAD-FMK (50 μM; TCZ), a necroptosis inducer, were used as positive control. MFI, mean fluorescence intensity. c is a summary plot; d shows representative imaging flow cytometry plots. a Data are representative of two independent experiments with all inhibitors using DC derived from different donors. Additional experiments employing combinations of Z-VAD-FMK and one or two of the other inhibitors were repeated 3–8 times using cells from different donors. n = 3 for experimental groups and for mock-infected controls. b, c Data are representative of two independent experiments using cells from different donors. n = 3 for experimental groups and for controls. ***p < 0.001, ANOVA followed by Tukey’s HSD test, with an additional Bonferroni multiple-testing correction for summarized data. Values shown are median ± s.e.m