Fig. 7.

Model validation: IFNβ pre-stimulation increases the fraction of responding cells and response strength. Following 24 h of quiescence (a, b) or pre-stimulation with 1000 U/ml IFNβ (a, d), WT MEFs were stimulated with 1 μg/ml poly(I:C) for 0–24 h, or they were only stimulated with 1000 U/ml IFNβ for 0–24 h (c). a Protein levels of model components, characterised by western blotting and numerical model simulations. Whole-cell extracts were analysed with antibodies against NF-κB–IRF3–STAT1/2 pathways components. (*) = IKK isoform-dependent phosphorylation sites: p-IKKα Ser176/180, p-IKKβ Ser 177/181. Representative experiments out of 2 are shown. Trajectories show averages of over 200 independent stochastic simulations; the colour key is located next to protein labels. b–d Cells were fixed and immunostained for (b, d) RelA (NF-κB) and IRF3 or RelA and IκBα, or (c) p-STAT (Tyr701). Representative excerpts from confocal images show cells at the indicated times after stimulation. c, d Histograms (n ≥ 600, from one out of 2 experiments) show the full time course of translocation, as defined for Fig. 2. Scale bars: 50 μm. e, f Following 24 h of quiescence or pre-stimulation with 1000 U/ml IFNβ, WT and Stat1^–/– MEFs were stimulated with 1 μg/ml poly(I:C) for 0–24 h, fixed and stained with antibodies against RelA and IRF3. e Scatter plots show nuclear translocations of RelA vs. IRF3 (as defined for Fig. 2b, n = 300); ρ is the Pearson correlation coefficient. f Fractions of active cells (see Fig. 5d) calculated from the scatter plots in e. See Supplementary Data 5, 6 and 7, 8 for uncropped immunostaining images corresponding to c, d and scatter plots for Stat1^–/– MEFs in e