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. 2010 Oct 30;409(2):175–188. doi: 10.1016/j.virol.2010.10.008

Fig. 1.

Fig. 1

Characterization of Huh7.5 cells stably expressing human ISG20 or its catalytically inactive mutant (ISG20m). (A) Immunofluorescence staining of ISG20 expression (using anti-myc mAb) in Huh7.5 cells mock-transfected (upper panels) or transiently transfected with a vector encoding myc-tagged ISG20 (lower panels). DAPI staining of nuclei was shown in the right panels. Similar subcellular localization of ISG20 was observed in Huh7.5 cells stably transduced with human ISG20 (7.5-ISG20) or the D94G mutant ISG20 (7.5-ISG20m) (data not shown). (B) Immunoblot analysis of ISG20 expression in 7.5-ISG20, 7.5-ISG20m and cells stably expressing the empty vector (Bsr). ISG20/ISG20m was probed with anti-myc mAb (upper panel) and ISG20 (lower panel), respectively. Actin was detected with mAb anti-actin (upper panel), to demonstrate equal sample loading. Note that ISG20m expressed to a slightly higher level than ISG20. It is unknown whether this reflects an auto-regulation of its own expression by ISG20. (C) Realtime PCR analysis of ISG20 mRNA levels in Huh7 and 7.5-ISG20 cells mock-treated or stimulated with IFN-α (500 U/ml) or infected with SeV (100 HAU/ml) for 16 h. mRNA abundances were normalized to cellular 28S rRNA. Fold differences were calculated by dividing normalized mRNA abundance following various treatments by that of the mock-treated Huh7 cells. IFN-α induced endogenous ISG20 expression by 7- to 15-fold in Huh7 and 7.5-ISG20 cells, while SeV upregulated ISG20 mRNA by 3-fold in Huh7 cells. (D) Triplicate wells of cells in 24-well plates were cultured for the indicated time periods and cell proliferation assessed by MTT assay. (E) ISG20 or ISG20m expression does not reconstitute the RIG-I signaling defect in Huh7.5 cells. 7.5-Bsr, 7.5-ISG20, 7.5-ISG20m, and normal Huh7 cells were mock infected or infected with 100 HAU/mL of SeV for 16 h prior to cell lysis and immunoblot analysis of ISG56, SeV and ISG20 (using anti-myc tag mAb). Asterisks in ISG56 panel denote nonspecific bands. Huh7.5 cells treated with recombinant human IFN-α for 16 h (lane 1) was included as a positive control for ISG56 expression.