FIG 5.

Roles of endogenous IFN-β and Stat1 in the activity of the cytokine cluster. (A) Huh-7 cells were treated with selected cytokines (1 nM) for 6 h. Quantitative RT-PCR using TaqMan primers specific for human IFN-β (and GAPDH as a control) was performed. (B) Time-dependent expression of IFN-β mRNA. Huh-7 cells were treated with IL-17F, BTC, and IFNL2 (1 nM) for the indicated times. Quantitative RT-PCR using TaqMan primers specific for human IFN-β and GAPDH was performed. (C) Wild-type (WT) and U3A (Stat1-deficient) fibroblasts were treated with the selected cytokine panel (1 nM) for 6 h. Quantitative RT-PCR using TaqMan primers specific for human IFN-β was performed, and the results were normalized to human GAPDH levels. (D) WT and U3A (Stat1-deficient) cells were treated with a selected cytokine panel (1 nM) for 6 h. Quantitative RT-PCR using TaqMan primers specific for human IFIT3 was performed, and the results were normalized to human GAPDH levels. (E) Huh-7 cells were transfected with nanoluciferase (NanoLuc) reporter fused to a minimal IFNB1 promoter and then subjected to the indicated IFN and cytokines for 6 h. Cells were lysed, and luciferase activity was quantitated and presented as fold increase over the control (no cytokine treatment). The data represent means and SEM of triplicate readings from two independent experiments. (F) Huh-7 cells were transfected with nanoluciferase reporter fused to NF-κB response elements and then subjected to the indicated IFN and cytokines for 6 h. The cells were lysed, and the luciferase activity was quantitated and presented as fold increase over the control (no cytokine treatment). (G) Huh-7 cells were pretreated with 10 μM NF-κB inhibitor or DMSO for 1 h before adding 1 nM each of the cytokines and incubating for a further 6 h. Quantitative RT-PCR using TaqMan primers specific for human IFN-β (and GAPDH as a control) was performed. The data are means and SEM from two independent experiments. *, P < 0.01; **, P < 0.001.