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A, B
SV40 large T antigen‐immortalised MEFs of the indicated genotypes were treated or not with 100 U/ml of IFN‐A/D for 16 h. (A) Cell extracts were subjected to Western blot analysis using the indicated antibodies. Asterisk (*) indicates non‐specific bands. (B) Cells were treated with 30 ng/ml TNF and 20 μM zVAD (TZ) or were infected with the indicated viruses at an MOI of 10. After 16 h, cell viability was assessed using CellTiter‐Glo reagent. Values for untreated cells were set to 100%.
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C
HEK293T cells were transfected with 50 ng NF‐κB firefly luciferase and 25 ng Renilla luciferase reporter plasmids, together with an expression vector for RIPK3. Luciferase activity was measured after 24 h, and the ratio of firefly and Renilla luciferase was set to 1 for control cells that did not receive RIPK3 plasmid.
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D
NIH3T3 cells were treated with IFN‐A/D for 16 h, and cell extracts were analysed by Western blot (top). Asterisk (*) indicates a non‐specific band. ZBP1‐3xFLAG‐reconstituted NIH3T3 cells were also tested by Western blot (bottom).
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E
ZBP1‐reconstituted NIH3T3 cells were infected as indicated and analysed as in (B).
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F
Cells were treated with 1,000 U/ml of IFN‐A/D for 16 h, and cell extracts were analysed by Western blot. Arrows indicate endogenous (lower band) and exogenous 3xFLAG‐tagged ZBP1 (upper band).
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G
Cells were infected with MCMV‐M45mutRHIM at an MOI of 10 or treated with TZ and analysed as in (B).
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H
Cell death was monitored upon infection or TZ treatment using an in‐incubator imaging platform (Incucyte) and the dye YOYO‐3, which stains dead cells.
Data information: Data are representative of three or more independent experiments. Panels (B, C, E, G and H) represent mean ± SD (
= 3). *
< 0.001; two‐way ANOVA.
