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. 2017 Sep 20;7:11988. doi: 10.1038/s41598-017-12154-4

Figure 6.

Figure 6

Hypo-responsiveness is mediated by IRAK-1 and can be mimicked by its knockdown. (a,b) A549 cells were stimulated with IL-1β (1 ng/mL, 2 h) followed by medium renewal (4 h incubation) and flagellin stimulation (100 ng/mL, 3 h). (a) Protein expression was analysed by western blotting and quantified. One out of four representative blots is shown. (b) The experimental data were used to validate model predictions. (c,d) THP-1 cells were stimulated with L. pneumophila (MOI 0.25 or 0.5) for 48 h. Upon their removal and subsequent medium renewal (4 h incubation), A549 cells were stimulated with L. pneumophila (MOI 100, 3 h). (c) Protein levels of IRAK-1 and α tubulin were analysed by western blotting and quantified. One out of four representative blots is shown. (d) Model predictions were validated using experimental data. (e,f) A549 cells were transfected with control (scr) or IRAK-1 siRNA (48 h) and stimulated with flagellin (100 ng/mL) or TNF-α (100 ng/mL) for 3 h. (e) The model was used to predict IL-8 expression upon IRAK-1 depletion, which was in accordance with the experimental data. (f) IL-8 expression was analysed by RT-qPCR and normalized to an untreated scr control (mean ± SEM, n = 4). (e) The data were compared with model predictions. Statistical analysis: (f) 2-Way ANOVA with Sidak’s multiple comparison test as described; *Compared to corresponding scr, ***p ≤ 0.001; ns = not significant.