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. 2017 Jun 19;7:3821. doi: 10.1038/s41598-017-04186-7

Figure 5.

Figure 5

IFN-α unresponsiveness depends on ISG15 and USP18 in cells that overexpress IFN-λ4 or treated with IFN-λ4. (A) PHHs with the IFNL4 ΔG/ΔG genotype were infected with JFH1 HCVcc at 5 MOI. After 72 hours, the cells were treated with 100 IU/ml of IFN-α for 30 minutes. (B) Huh-7 cells were transfected with control or IFN-λ4-expressing plasmids. After 72 hours, the cells were treated with 100 IU IFN-α for 15 minutes. The cells were harvested, and protein expression was analysed by immunoblotting. (C) Huh-7 cells were transfected with control, USP18-, or ISG15-targeting siRNAs. After 24 hours, the cells were transfected with control or IFN-λ4-expressing plasmids. After 48 hours, the cells were re-transfected with the respective siRNAs. The cells were harvested, and gene expression was analysed by immunoblotting 24 hours after the second siRNA transfection. (D,E) Huh-7 cells were transfected with control, USP18-, or ISG15-targeting siRNAs. After 24 hours, the cells were transfected with control or IFN-λ4-expressing plasmids. After 48 hours, the cells were re-transfected with the respective siRNAs. After 48 hours, the cells were treated with 100 IU/ml of IFN-α for 15 minutes (D) or for 6 hours (E). Then the cells were harvested, and protein and gene expression were analysed by immunoblotting (D) or real-time qPCR (E). All the data are representative of two independent experiments. (F) Huh-7 cells were treated with or without 100 ng/mL recombinant IFN-λ4. After 48 hours, the cells were transfected with control siRNAs, USP18-, or ISG15-targeting siRNAs. The USP18-, or ISG15-targeting siRNAs used in this experiment had different sequences from siRNAs used in the experiment for (D). After 48 hours, the cells were treated with 100 IU/ml of IFN-α for 15 minutes. Then the cells were harvested, and protein expression were analysed by immunoblotting. (G) PHHs with IFNL4 ΔG/ΔG genotype were infected with JFH1 HCVcc at 15 MOI. After 6 hours, the cells were treated with 10 μg/ml anti-IFN-λ1 and 10 μg/ml anti-IFN-λ2/3 or IgG for 72 hours. The cells were harvested, and ISG15 gene expression was analysed by real-time qPCR. Data are presented as means ± S.E.M. **P ≤ 0.01, ***P ≤ 0.001 (Student’s t-test).