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. 2020 Sep 24;11:575818. doi: 10.3389/fimmu.2020.575818

FIGURE 1.

FIGURE 1

G10 elicits a type I IFN response in porcine cells. (A) Schematic representation of the STING-mediated type I IFN response. (B) WT, Sting–/–, Tbk1–/–, Irf3–/–, and Ifnar1–/– PK15 cells were seeded in 12-well plates at a density of 1 × 105 per well. On the next day, cells were treated with G10 at the indicated concentrations for 24 h. Total mRNA was then reverse-transcribed to cDNA and IFN-β mRNA was assessed by RT-qPCR analysis. The results were normalized to the level of β-actin expression. *P < 0.05, **P < 0.01, ***P < 0.001 determined by two-tailed Student’s t-test. (C) WT, Sting–/–, and Ifnar1–/– 3D4/21 cells were seeded in 12-well plates at a density of 1 × 105 per well. On the next day, cells were treated as in B. IFN-β mRNA was assessed by RT-qPCR analysis. The results were normalized to the level of β-actin expression. *P < 0.05, **P < 0.01, ***P < 0.001 determined by two-tailed Student’s t-test. (D) WT and Sting–/– PK15 and 3D4/21 cells were seeded in 12-well plates at a density of 1 × 105 per well. On the next day, cells were treated as in B. The medium was then harvested and IFN-β secretion was quantified by ELISA. *P < 0.05, **P < 0.01, ***P < 0.001 determined by two-tailed Student’s t-test. (E) WT, Sting–/–, Tbk1–/–, Irf3–/–, and Ifnar1–/– PK15 cells were seeded in 12-well plates at a density of 1 × 105 per well. On the next day, cells were treated as in B. ISG15 mRNA was assessed by RT-qPCR analysis. The results were normalized to the level of β-actin expression. *P < 0.05, **P < 0.01, ***P < 0.001 determined by two-tailed Student’s t-test. (F) WT, Sting–/–, and Ifnar1–/– 3D4/21 cells were seeded in 12-well plates at a density of 1 × 105 per well. On the next day, cells were treated as in B. ISG15 mRNA was assessed by RT-qPCR analysis. The results were normalized to the level of β-actin expression. *P < 0.05, **P < 0.01, ***P < 0.001 determined by two-tailed Student’s t-test. (G) WT and Sting–/– PK15 and 3D4/21 cells were seeded in 12-well plates at a density of 1 × 105 per well. On the next day, cells were infected with PRV-QXX (MOI = 1) and simultaneously treated with G10 as in B. Virus was harvested by three freeze–thaw cycles and PRV titer was assessed with TCID50 assays. *P < 0.05, **P < 0.01, ***P < 0.001 determined by one-way ANOVA.