Figure 3. Caspase-6 Promotes IAV-Induced Pyroptosis, Apoptosis, and Necroptosis.

(A) Real-time analysis of cell death in bone-marrow-derived macrophages (BMDMs) using the IncuCyte imaging system and SYTOX Green nucleic acid staining after infection with influenza A virus (IAV) for 12 h. The original magnification is ×20. The red denotes the cells counted as dead in the analysis.

(B) Quantification of the cell death observed in (A).

(C) Immunoblot analysis of caspase-6 (CASP6) in mouse embryonic fibroblasts (MEFs) following CRISPR-directed deletion.

(D) Microscopic analysis of cell death in MEFs infected with IAV for 24 h. The original magnification is ×10.

(E) Quantification of the cell death observed in (D).

(F) Immunoblot analysis of the pro-and cleaved forms of caspase-3 (CASP3), caspase-7 (CASP7), and gasdermin D (GSDMD) in BMDMs after IAV infection for 9 h. Actin is used as the internal control.

(G) Immunoblot analysis of the pro- and cleaved forms of CASP3 and CASP7 in MEFs after IAV infection for 24 h. Actin is used as the internal control.

(H) Immunoblot analysis of pro- and cleaved caspase-8 (CASP8) in BMDMs after IAV infection for 9 h. Actin is used as the internal control.

(I) Immunoblot analysis of pro- and cleaved CASP8 in MEFs after IAV infection for 24 h. Actin is used as the internal control.

(J) Immunoblot analysis of phosphorylated mixed lineage kinase domain-line (pMLKL) and total MLKL (tMLKL) in BMDMs after IAV infection at the indicated time points. Actin is used as the internal control.

****p < 0.0001. Analysis was performed using the Student’s t test (B) or one-way ANOVA (E). Data are shown as mean ± SEM (B and E). See also Figure S2.