Fig. 5

USP18 upregulation inhibited IFN-induced Jak/STAT signaling pathway. HepAD38 cells and HepG2 cells were seeded in 6-well plate without any treatment, respectively. Forty-eight hours later, supernatant IFNα (a, left) and IFNβ (a, right) and intracellular mRNA expression of ISGs (b) were analyzed by ELISA assay and real-time PCR, respectively. To investigate the effects of USP18 on STAT phosphorylation, HepAD38 cells were transfected with WT-USP18, USP18-C64S or MOCK for 48 h and treated with 500 IU/ml IFNα for 30 min before harvested. Western blot was used to analyze the expression of STAT1 and p-STAT1 (c, left). The interferon stimulated response element (ISRE) activity was quantified by dual luciferase reporter gene assay. Briefly, HepAD38 cells were co-transfected with WT-USP18, USP18-C64S or MOCK and ISRE-luc reporter plasmid /pRL-TK reporter plasmid for 24 h, and then left untreated or treated with 100 IU/ml or 1000 IU/ml IFNα for 24 h before the cells were lysed (c, middle). HepAD38 cells were transfected with WT-USP18, USP18-C64S or MOCK for 48 h and treated with 500 IU/ml IFNα for additional 24 h. Expression of ISGs mRNA including MxA and OAS2 were detected by real-time PCR (c, right). WT-USP18, wide type USP18; MOCK, empty plasmid. Results are presented as means ± SD (n ≥ 3). *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001