Fig 2: FLAG-Iso1 overexpression augments the cellular response to IFNL3. (A) Quantitation of percent pSTAT1+ in HEK293T-EV and FLAG-Iso1 cells after 0, 1, and 4 h of IFNL3 treatment (100 ng/mL) (representative data, two independent experiments). (B) HEK293T-EV and FLAG-Iso1 cells were co-transfected with plasmids encoding Firefly Luciferase under control of an ISRE promoter and Renilla Luciferase under control of a CMV promoter. Cells were treated ±dox (100 ng/mL) for 24 h prior to mock or IFNL3 (100 ng/mL) treatment for 24 h and harvested for dual luciferase assay (representative data, three independent experiments). (C) HEK293T-EV and FLAG-Iso1 cells were treated ±dox (100 ng/mL) for 24 h prior to IFNL3 treatment for 24 h (0, 1, 10, or 100 ng/mL), then harvested for qRT-PCR analysis of VIPERIN and GAPDH (representative data, four independent experiments). Statistical significance represented by lone asterisks reflect comparisons between identically treated EV and FLAG-Iso1 cells. Statistical significance represented by bars and asterisks reflect comparisons between -dox and +dox conditions within each cell line. Error bars represent standard error of the mean. * = p < 0.05, ** = p < 0.01, *** = p < 0.001. NS = not significant.

Fig 3: FLAG-Iso2 overexpression differentially influences the cellular response to IFNL3 based on receptor abundance. (A) pSTAT1 (two independent experiments), (B) ISRE activity (five independent experiments), and (C) VIPERIN expression (three independent experiments) were analyzed as described in Figure 3. Statistical significance represented by lone asterisks reflect comparisons between identically treated EV and FLAG-Iso2 cells. Statistical significance represented by bars and asterisks reflect comparisons between −dox and +dox conditions within each cell line. Error bars represent standard error of the mean. * = p < 0.05, ** = p < 0.01, *** = p < 0.001. NS = not significant.

Fig 4: Relative expression of FLAG-IFNLR1 isoforms differentially influences IFNL3-dependent gene expression. HEK293T WT and stable lines were induced ±dox (100 ng/mL) for 24 h prior to treatment ±IFNL3 (100 ng/mL) for an additional 24 h. RNA was collected and gene counts were quantitated using NanoString analysis (nCounter Human Immunology v2 Panel). Data are shown as log transformed normalized counts of differentially expressed genes (>2-fold change in any condition).

Fig 5: The cellular response to IFNL3 is inversely proportional to FLAG-Iso2 abundance. Two independent FLAG-Iso2 stable lines were transfected with CMV-Renilla and ISRE-Firefly plasmids and dox-treated (dose range between 0–100 ng/mL) for 24 h. Cells were then treated with IFNL3 (100 ng/mL) for 24 h prior to dual luciferase assay. (A) FLAG-Iso2 clonal line characterized in Figure 4 and (B) an additional independent FLAG-Iso2 clonal line (representative data, two independent experiments). Statistical analysis compares IFNL3 relative to mock-treated cells at a given dox concentration. Error bars represent standard error of the mean. * = p < 0.05, ** = p < 0.01, *** = p < 0.001. NS = not significant.