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. 2020 Mar 2;23(3):100958. doi: 10.1016/j.isci.2020.100958

Figure 2.

Figure 2

Human and Bat Cells Expressing IRF3-S185 Display Enhanced Antiviral Protection

(A) Schematic representation of the experimental strategy. IRF3 knockout (KO) bat and human cells were transfected with varying concentrations of wild-type (WT) or altered IRF3 expression plasmids for 24 h. The cells were then stimulated with poly(I:C) for 6 h, followed by infection with vesicular stomatitis virus (VSV) that was engineered to express green fluorescent protein (GFP). Nineteen hours after infection, GFP expression was measured as a surrogate for virus replication.

(B) VSV-GFP replication in E. fuscus IRF3 KO kidney cells (cr3-8) transfected with varying concentrations of plasmids expressing WT (S185) or altered (L185) E. fuscus IRF3 and mock treated or treated with poly(I:C) (n = 3). No plasmid and 200 ng of empty vector were used as transfection controls. Immunoblots: IRF3 protein levels in cr3-8 cells mock transfected, transfected with 200 ng empty vector (pcDNA), or transfected with varying concentrations of WT (S185) or altered (L185) IRF3 expression plasmids.

(C) VSV-GFP replication in P. alecto IRF3 KO kidney cells (PakiT03-4G) transfected with varying concentrations of plasmids expressing WT (S185) or altered (L185) P. alecto IRF3 and mock treated or treated with poly(I:C) (n = 3). No plasmid and 200 ng of empty vector were used as transfection controls. Immunoblots: IRF3 protein levels in PakiT03-4G cells mock transfected, transfected with 200 ng empty vector (pcDNA) or transfected with varying concentrations of WT (S185) or altered (L185) IRF3 expression plasmids.

(D) VSV-GFP replication in human IRF3 KO cells (THF-IRF3-KO) transfected with varying concentrations of plasmids expressing WT (L185) or altered (S185) human IRF3 and mock treated or treated with poly(I:C) (n = 3). No plasmid and 200 ng of empty vector were used as transfection controls. Immunoblots: IRF3 protein levels in THF IRF3 KO cells mock transfected, transfected with 200 ng empty vector (pcDNA) or transfected with varying concentrations of WT (L185) or altered (S185) IRF3 expression plasmids.

Data are represented as mean ± SD, n = 3, ∗∗p < 0.01, ∗∗∗p < 0.001 (Student's t test). GFP expression is represented after normalization with mock infected cells. IRF3 protein expression and quantification data are expressed as a ratio of IRF3/GAPDH levels on top of the blots. Blots were quantified using Image Studio (LI-COR) (n = 3). KO, knockout; WT, wild-type; Ef, E. fuscus; Pa, P. alecto; Hu, human; NS, not significant.