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. Author manuscript; available in PMC: 2017 Jul 13.
Published in final edited form as: Cell Host Microbe. 2016 Jun 16;20(1):13–24. doi: 10.1016/j.chom.2016.05.011

Figure 3. RIPK3 activates parallel pathways of necroptosis and apoptosis upon IAV infection.

Figure 3

(A) Ripk3+/+ MEFs were infected with PR8 (m.o.i.=2) in the presence or absence of zVAD (50µM), RIPK1 kinase inhibitors [GSK’963 (5µM), GSK’481 (5µM), and Nec-1 (50µM)], or RIPK3 kinase inhibitors [GSK’872 (5µM), and GSK’843 (5µM)] and cell viability was determined 24 h.p.i. TCZ-treated wild-type MEFs exposed to the same doses of RIPK1 or RIPK3 inhibitors were used as controls. (B) LET1 lung epithelial cells were infected with PR8 in the presence or absence of zVAD (50µM) or GSK’843 (5µM), and viability was determined 12 h.p.i. LET1 cells treated with TCZ were included as controls. (C) Human HT-29 cells were infected with PR8 (m.o.i.=5) in the presence or absence zVAD (50µM) or GSK’840 (3µM) and viability was determined 14 h.p.i. HT-29 cells treated with human TNF-α (0.5ng/mL), SMAC mimetic LCL161 (5µM), and zVAD (50µM) were included as controls (TSZ). (D) Kinetics of cell death after wild-type or UV-inactivated (PR8 UV) PR8 infection in ripk3+/+ MEFs in the presence or absence of caspase inhibitor QvD (40µM) or GSK’872 (5µM) was analyzed for up to 36 h.p.i. Cell death was determined Sytox Green uptake, and quantified as number of Sytox Green positive cells over time. (E) Wild-type MEFs infected with PR8 in the presence of GSK’843 (5µM), zVAD (50µM) or both inhibitors together were examined for phosphorylated MLKL or cleaved caspase 8 by immunoblot analysis at the indicated times p.i. Error bars represent mean +/− S.D. * p <0.05; ** p <0.005. See also Figs. S4 and S6.

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