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. 2020 Feb 14;94(5):e01535-19. doi: 10.1128/JVI.01535-19

FIG 2.

FIG 2

The IFNL4 promoter is not activated by the same stimuli that readily activate the IFNL1 promoter. (A) The promoter regions of the human IFNL1 and IFNL4 genes corresponding to 2,186 and 2,387 bp, respectively, upstream of their translation start site were inserted in front of the firefly luciferase coding sequence in the pGL3.1-Basic plasmid. (B) HEK293T cells were cotransfected with a pGL3.1 plasmid containing the IFNL1 or IFNL4 promoter, a plasmid constitutively expressing the Renilla luciferase gene, and increasing amounts of a plasmid expressing MAVS. At 24 h posttransfection, firefly/Renilla luciferase activity was quantified. The experiment was performed in biological triplicates and the data are presented as a bar chart and scatter plot with mean ± SD (n = 3). (C) HEK293T cells were cotransfected with a pGL3.1 plasmid containing the IFNL1 or IFNL4 promoter and a plasmid constitutively expressing the Renilla luciferase gene. At 24 h posttransfection, the cells were infected with SeV for another 24 h before quantification of firefly/Renilla luciferase activity. One representative out of two independent experiments is shown, each with biological triplicates. The data are presented as a bar chart and scatter plot with mean ± SD (n = 3). (D and E) IRF3-knockout HEK293T cells were cotransfected with a pGL3.1 plasmid containing the IFNL1 (D) or IFNL4 (E) promoter in front of a firefly luciferase gene, a plasmid constitutively expressing the Renilla luciferase gene, and a plasmid constitutively expressing IRF1, IRF2, IRF3, IRF4, IRF5, IRF6, IRF7, or IRF8. At 24 h posttransfection, the cells were infected with SeV for another 24 h before quantification of firefly/Renilla luciferase activity. One representative out of two independent experiments is shown, each with biological triplicates. The data are presented as a bar chart and scatter plot with mean ± SD (n = 3). Statistical significance was determined using an unpaired t test. ****, P < 0.0001; ***, 0.0001 < P < 0.001; **, 0.001 < P < 0.01; *, 0.01 < P < 0.05; ns, P ≥ 0.05.