Figure 2A-B qRT-PCR detection for the mRNA expression of Ifnb1 (A), Ifna4, Cxcl10 and Ccl5 (B) in WT (n=3) and Akt2 KO (n=3) PEMs stimulated with lipo-poly(I:C), VSV, lipo-poly(A:T), lipo-ISD or HSV-1 for 6 hours. C The expression of Ifnb1 mRNA level by qRT-PCR in WT (n=3) and Akt2 KO (n=3) PEMs treated with poly(I:C) (10 μg/mL) for 6 hours (left panel) or LPS (1 μg/mL) for 2 hours (right panel). Data information: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 and ns, not significant (P > 0.05); using two-way ANOVA test Data are from three independent experiments Error bars mean ± SEM D The expression of Ifnb1 in WT (n=3) and Akt2 KO (n=3) primary-MEFs with VSV treatment (MOI, 0.1) for 6 hours was measured by qRT-PCR (left panel). After VSV treatment for 3 hours, the culture suspension was discarded and wash 3 times with PBS, then primary-MEFs was cultured with fresh medium for another 15 hours for analysis of the VSV-infected (GFP) primary-MEFs by fluorescence microscope (middle panel, representative images) and by FACS assay (right panel). Bar, 100 μm. Data information: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 and ns, not significant (P > 0.05); using two-way ANOVA test Data are from three independent experiments or representative of three independent biological replicates Error bars mean ± SEM E MEF (left panel, n=3) and HEK293T cells (right panel, n=3) were transfected with AKT2 for 24 hours and stimulated with VSV for 6 hours, then cells were harvested for detection the mRNA expression of Ifnb1 by qRT-PCR. Data information: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 and ns, not significant (P > 0.05); using a one-way ANOVA test Data are from three independent experiments Error bars mean ± SEM F IFNβ1 luciferase assays of HEK293T cells with transfection of AKT2/AKT2-T309A/S474A and TBK1 (n=3) or infection of VSV (n=3). Data information: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 and ns, not significant (P > 0.05); using a one-way ANOVA test Data are from three independent experiments Error bars , mean ± SEM G Zebrafish larva at 48 hour after fertilization were micro-injected GFP-fused VSV (1 × 103 PFU/larvae) for 18 hours, then the representative images of VSV-infected zebrafish larva were collected by fluorescence microscope. The infected area (GFP) and macrophages (Red) are indicated by arrows. Bars, 200 μm. Data information: * Data are from three independent biological replicates H Zebrafish larvae were overexpressed the indicated protein for 48 hours and challenged with VSV for another 6 hours, then the mRNA levels of ifn1 in zebrafish larvae were measured by qRT-PCR (left panel). Every dot represents three zebrafish embryos. Horizontal square bracket shows the statistical analysis of the comparison with 'PBS mock', the rest shows the comparison with 'PBS VSV'. PBS mock, n=5; PBS VSV, n=13; AKT2 VSV, n=7; AKT2-T309A/S474A VSV, n=9. H&E staining (middle panel) and survival rates (Kaplan-Meier curve) (right panel) were collected from zebrafish larvae after VSV micro-injection for 18 hours or longer. The arrows indicated the VSV-infected eye and skeletal muscle in zebrafish larvae. Bars, 100 μm. Data information: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 and ns, not significant (P > 0.05); using a one-way ANOVA test or log-rank (Mantel-Cox) test Data are from three independent biological replicates Error bars mean ± SD).