Fig. 6.

Increased levels of ISG15 and USP18 in cells after prolonged stimulation with IFN-λ₃ or in HCV-infected livers. (A and B) Huh-7.5 cells were treated with 10 ng/mL IFN-λ₃ for 5 d and rested for 16 h. Then, 100 IU of IFN-α or peg–IFN-α2b was added. After 30 min, immunoblotting was performed for the detection of STAT1, PY-STAT1, STAT2, and IRF9 (A). After peg–IFN-α2b treatment for 6 or 24 h, the expression of ISGs was examined by TaqMan real-time quantitative PCR (B). (C and D) Huh-7.5 cells were treated with 10 ng/mL IFN-λ₃ for 5 d and rested for 16 h. Immunoblotting of ISG15 and USP18 was performed (C), and the mRNA levels of ISG15 and USP18 were examined by TaqMan real-time quantitative PCR (D). (E) Huh-7.5 cells were treated with 10 ng/mL IFN-λ₃. After 5 d, siRNAs targeting USP18 or ISG15 were introduced by transfection. Forty-eight hours after transfection, cells were harvested, and immunoblotting of ISG15 and USP18 was performed. (F–H) Immunoblotting of ISG15 and USP18 was performed in control livers without viral hepatitis (n = 4) and HCV-infected livers (n = 8). Bands for STAT1, ISG15 and USP18 are presented (F). The relative band intensities (% of actin) are presented as scatter plots (G). Correlation between the band intensities of ISG15 and those of USP18 is presented (H). The data represent the means ± SEM (n = 3). *P < 0.05, **P < 0.01 compared with control.