Figure 1. RIPK3 is required for IAV-induced lysis of MEFs and alveolar epithelial cells.

(A) Ripk3^+/+ and ripk3^−/− MEFs were infected with the indicated strains of influenza virus at m.o.i.=2 or treated with TNF-α (50ng/ml) in the presence of cycloheximide (250ng/ml) and zVAD (50µM) and cell viability was determined at 24 h.p.i. (B) Photomicrographs of ripk3^+/+ and ripk3^−/− MEFs infected with PR8 or treated with TCZ for 24 h. (C) FACS analysis of ripk3^+/+ and ripk3^−/− MEFs infected with PR8-GFP (m.o.i.=2). The y-axis shows side scatter. (D) Ripk3^+/+ and ripk3^−/− MEFs infected with PR8 were examined for virus replication by immunoblotting with antiserum raised against PR8 or a monoclonal antibody to NS1. A non-specific band detected in uninfected lysates by the anti-PR8 antiserum is indicated with an asterisk (*). Molecular weights (in kDa) are shown to the left. (E) Kinetics of cell death after PR8 infection of ripk3^+/+ and ripk3^−/− MEFs at the indicated m.o.i.s. (F) Ripk3^−/− (in the presence or absence of 50µM zVAD), ripk3^−/−casp8^−/−, or ripk3^−/−fadd^−/− MEFs were infected with PR8 and cell viability was determined 36 h.p.i. (G) Parental LET1 lung epithelial cells, or LET1 cells in which RIPK3 expression was ablated by CRISPR/Cas9 targeting of the sequence 5’-TGAGAACGTTCTGCTCCTGC-3’ in the murine ripk3 gene, were infected with PR8 and viability (left) or progeny virion output (right) was determined 12 h.p.i. Inset: Immunoblot showing RIPK3 levels in these cells, with β-actin included as a loading control. Error bars represent mean +/− S.D. * p <0.05; ** p <0.005. See also Figs. S1, S2 and S6.