Fig. 8

Interferon response is not affected by the formation of viral inclusions. a Stable cell lines expressing GFP-Rab11 WT or GFP-Rab11 DN were infected or mock-infected (M), at an MOI of 3, with PR8 WT or NS1-N81 viruses. a Cells were fixed at 8 and 16 hpi and stained for NP (in red). Bar = 10 μm. b The frequency distribution of NP inclusions within the three area categories (in μm²) was plotted for each cell line. Statistical analysis of data was performed using a non-parametric Kruskal–Wallis test, followed by Dunn’s multiple comparisons test (*** p < 0.001 for GFP-Rab11 WT cells; no statistical significance found for GFP-Rab11 DN cells). Statistical analysis compares the area of all inclusions between conditions. An average of 30 cells was analyzed per condition. A single experiment representative of two independent experiments is shown. c Expression of IFN-β, IFN-α, IL-29 and viperin was evaluated at the level of transcription by RT-qPCR in relation to GAPDH. Poly(I:C) was used as a positive control for maximum expression of these transcripts. Statistical analysis of data was performed using two-way ANOVA test, followed by Sidak multiple comparisons test (no statistical significance between conditions found). Data represents the average of three independent experiments. d Expression of phosphorylated IRF3, GFP, NP, NS1, and GAPDH was evaluated at the protein level by western blotting. e The levels of secreted IFN-β were quantified by ELISA in cell supernatants at 24 hpi. Poly(I:C) was used as a positive control for maximum expression of IFN-β protein. The limit of detection of this method is 30 pg mL⁻¹ (dashed line). Statistical analysis of data was performed using two-way ANOVA test, followed by Sidak multiple comparisons test (no statistical significance between conditions found). Data represent the average of three independent experiments. f At the indicated times, supernatants were collected and viral production was evaluated by plaque assays using MDCK cells. Statistical analysis of data was performed using Holm–Sidak multiple comparisons test (**p < 0.01, ***p < 0.001). Data correspond to one representative experiment out of three independent experiments