Fig. 3.

Dectin-1 and CARD9 are required for fungus and its PAMPs-induced necroptotic cell death. a Peritoneal macrophages were transfected with scrambled siRNA oligos, Dectin-1 siRNA oligos for 60 h. Knock-down efficiency was determined by detecting mRNA level through qPCR. b Peritoneal macrophages were transfected with the indicated siRNA oligos for 60 h and treated with zymosan or curdlan for 12 h. Cell viability was determined by measuring released LDH. c Immunoblot analysis of p-MLKL from peritoneal macrophages transfected with the indicated siRNA oligos for 60 h and then treated with zymosan, curdlan and zVAD for 3 h. d Densitometric quantification of p-MLKL/GAPDH relative level is shown from (c) . e Peritoneal macrophages were transfected with scrambled siRNA oligos, CARD9 siRNA oligos or RIPK3 siRNA oligos for 60 h. Knock-down efficiency was determined by detecting mRNA level through qPCR. f Peritoneal macrophages were transfected with the indicated siRNA oligos for 60 h and treated with zymosan or curdlan for 12 h. Cell viability was determined by measuring released LDH. g Peritoneal macrophages from Tnf^−/− mice were transfected with the indicated siRNA oligos for 60 h and treated with zymosan, curdlan and zVAD for 12 h. Cell viability was determined by measuring released LDH. h Immunoblot analysis of p-MLKL from Tnf^−/− peritoneal macrophages transfected with the indicated siRNA oligos for 60 h and then treated with zymosan, curdlan and zVAD for 3 h. i Densitometric quantification of p-MLKL/GAPDH relative level is shown from (h). Data are representative of three independent experiments. Error bars in (a, b, d–g, i) represent mean ± SEM. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001 by two-tailed test