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. 2021 Mar 25;12:1858. doi: 10.1038/s41467-021-22088-1

Fig. 6. MDA5-TBK1 signaling explains the innate inflammatory response to PVSRIPO.

Fig. 6

a, b MDMs were transfected with control siRNA or siRNA targeting MDA5 (48 h), followed by treatment with PVSRIPO, Poly(I:C), or LPS (48 h). a Immunoblot analysis of MDA5 depletion, viral protein 2 C, and other relevant proteins. b Cytokine release (top) and cell surface markers of activation (bottom) were measured. Fold-mock control siRNA cytokine concentrations were measured, geometric mean of %max induction values are presented; surface markers were normalized similarly, except baseline (mock control siRNA) %max values were subtracted and mean is shown. N = 6 experiments from two donors, asterisks indicate two-tailed Tukey’s post-hoc test p < 0.05 comparing siMDA5 to siCtrl values for each treatment (PVSRIPO: IFNα p < 0.0001, IFNβ p = 0.0001, IFNλ1 p < 0.0001, CXCL10 p = 0.0005, CD40 p = 0.0005, CD83 p = 0.006, PD-L1 p = 0.01, CCR7 p = 0.004; LPS TNF p < 0.0001). c MDMs were pre-treated (1 h) with DMSO, Bx795 (2 µM; TBK1:IKKε inhibitor), or IKK16 (400 nM: IKKα:β inhibitor) followed by treatment with PVSRIPO (MOI 10) or LPS (100 ng/ml). Supernatant cytokines were analyzed; IFNα/β were not induced by LPS and thus were not included in the heat map; values were normalized from four experiments (two donors) by setting DMSO treatment values to 100% for each cytokine; geometric mean is shown; asterisks denote Tukey’s post-hoc p < 0.05 (two-tailed) test vs DMSO control (*) or all groups (#) for each cytokine. See Supplementary Fig. 9 for extended data.