Journal: Journal of Virology
Article Title: ISG15 Modulates Type I Interferon Signaling and the Antiviral Response during Hepatitis E Virus Replication
Figure Lengend Snippet: ISG15 regulates type I IFN signaling. (A) Relative pSTAT1 levels. Huh7-S10-3 liver cells were transfected with 20 nM control siRNA (siCnt) or ISG15-siRNA (siISG15) or UBE1L-siRNA plus UBE2L6-siRNA (siE1E2). At 24 hpt, cells were treated with IFN-α (100 IU/ml) for 30 min to 7 h. Cell lysate (20 μg/lane) was analyzed by Western blotting with the indicated antibody, anti-pSTAT1 (1:1,000 dilution), anti-STAT1 (1:1,000 dilution), and anti-β-actin (1:1,000 dilution). Fold change in band intensity was determined using ImageJ software (NIH, Bethesda, MD). The data represent means ± SEM of results from five independent experiments. a, P ≤ 0.05; aa, P ≤ 0.01 (compared with siCnt plus IFN-α at the given time point). (B) IFN-stimulated response element (ISRE) promoter activity levels. Huh7-S10-3 liver cells were cotransfected with siCnt/siISG15/siE1E2 along with pGL4.45[luc2P/ISRE/Hygro] (firefly luciferase) and pGL4.74[hRluc/TK] (renilla luciferase). At 24 hpt, cells were treated with various concentrations of IFN-α. Relative levels of fold induction of the cell-associated firefly luciferase activity, compared to the corresponding untreated cell control levels, at 18 h post-IFN-α treatment were estimated using a dual-luciferase assay kit and were normalized with renilla luciferase expression levels. The data represent means ± SEM of results from triplicate sample experiments. aa, P ≤ 0.01 (compared to siCnt plus IFN-α). (C to F) ISG mRNA levels in siRNA-transfected and IFN-α-treated samples were measured for Mx1 (C), OAS1 (D), PKR (E), and ISG15 (F) using Sybr green qPCR. Fold change in mRNA levels compared to the untransfected control was calculated using the 2 −ΔΔ CT method, and the RPS18 gene was used as the housekeeping gene. The data represent means ± SEM of results from three independent experiments. a, P ≤ 0.05; aa, P ≤ 0.01; aaa, P ≤ 0.001 (compared to siCnt plus IFN-α).
Article Snippet: The mRNA levels of ISG15, OAS1, PKR, Mx1, USP18, and RPS18 (housekeeping control) were determined using SYBR master mix (Applied Biosystems, Grand Island, NY) with gene-specific primer sets ( ) and a Bio-Rad IQ5 system.
Techniques: Transfection, Western Blot, Software, Activity Assay, Luciferase, Expressing, SYBR Green Assay, Real-time Polymerase Chain Reaction