Fig. 2

IRF3 deficient primary microglia cultures show delayed downstream signaling and attenuated cytokine production on LPS challenge. A and C Representative images of western blots from primary microglia cultures treated with LPS. B Quantification of the westerns show significant IRF3 phosphorylation in the WT cells, following LPS stimulation. N = 6 for each group. Mann–Whitney test, **p < 0.01. D Quantification shows that 30 min after LPS stimulation, there is an increase in phosphorylation of NF-κB, p38, and ERK1/2 as indicated by the fold change > 1 over the 0-time point in WT and IRF3KO cultures. However, IRF3KO microglia cultures show significant reduction in the levels of phosphorylation compared to that of WT. N = 7,8 for each group. Mann–Whitney test, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. E Quantification shows that 120 min after LPS stimulation, there is an increase in phosphorylation of NF-κB, p38, and ERK1/2 as indicated by the fold change > 1 over the 0-time point for both genotypes. However, IRF3KO microglia cultures only show a significant reduction in the levels of phosphorylation of NF-κB, while those of p38 and ERK1/2 are indistinguishable from that of the WT. N = 7,8 for each group. Mann–Whitney test or unpaired t-test as appropriate. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. F Quantification of ELISA from cell culture supernatants shows that proinflammatory cytokines (IFNβ, TNFα, IL6, ILβ, IL1α) are significantly upregulated in WT microglia on LPS challenge. IRF3KO cultures show either no release or significantly reduced release of cytokines on LPS challenge. N = 4–7 for each group. Two-way ANOVA with Tukey’s multiple comparisons. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001