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HEP_23985_sm_SuppInfo-Figures.doc3547KSupplementary Figure 1: Upregulation of IRF7 mRNA levels with increasing doses and duration of treatment with ribavirin. (a) Huh7.5.1 cells were treated with ribavirin for 24 hours with increasing doses or with 100 μg/mL for increasing durations and harvested for qPCR determination of IRF7 levels. (b) qPCR was performed on the HepG2 cell line 24 hours after treatment with ribavirin at doses of 0, 10, and 100 μg/mL for IRF7, IRF9 and ISG15. Data is obtained from experiments performed in triplicate. Supplementary Figure 2: Induction of ISGs by interferon-α is greatest at 8 h. Huh7.5.1 cells were treated with 100 U/mL interferon-α and harvested at different time points for qPCR analysis of IP10, MX1, IFI44 and RSAD2 expression levels. Data is obtained from experiments performed in triplicate. Supplementary Figure 3: Synergistic induction of ISG15 by ribavirin and interferon-α. (a) qPCR analysis of ISG15 induction after treatment with ribavirin and interferon at various concentrations in Huh 7.5.1 cells. (b) Normalized isobologram demonstrating synergy between ribavirin (1, 3, 10, 30 μg/mL for 24 h) and interferon-α (1, 3, 10, 30, 100 U/mL for 8 h) on ISG15 mRNA levels for 14 combinations of the two drugs generated with the Calcusyn analysis software. (c) qPCR analysis of ISG15 and IRF9 induction after treatment with ribavirin (1, 3, 10 μg/mL for 24 h) and interferon-α (30 U/mL for 8 h) in Huh 7 cells. Data is obtained from experiments performed in triplicate. Supplementary Figure 4: Gene induction by ribavirin, interferon-α and poly(IC). qPCR analysis of (a) IFI44 and RSAD2 expression levels in Huh7.5.1 cells transfected with poly(IC) (6 μg/mL) at various time points. (b) Interferon-α (c) IFI44 and RSAD2 mRNA levels in Huh7.5.1 cells after treatment with ribavirin (100 μg/mL for 24 hours), interferon-α (100 U/mL for 8 hours) and poly(IC) (6 μg/mL for 8 hours). Data is obtained from experiments performed in triplicate. Supplementary Figure 5: Gene induction by ribavirin does not involve the TLR signaling pathway. qPCR analysis of (a) IRF7, IRF9, and ISG15 mRNA levels in Huh7.5.1 cells first treated with siRNAs for three days targeting TRIF (with quantification of siRNA mediated gene suppression) and (b) qPCR analysis of IRF7 in MYD88 siRNA treated cells (with quantification of siRNA mediated gene suppression) and then treated with or without ribavirin (100 mg/mL for 24 h). Data is obtained from experiments performed in triplicate. Supplementary Figure 6: Blocking Type I Interferon has no Effect on Gene Induction by Ribavirin. (a) qPCR analysis of IFN-α, IFN-β, IL28A/B, and IL29 following treatment with ribavirin (100 μg/mL for 24 h) and cycloheximide (10 μg/mL for 24h). Data is obtained from experiments performed in triplicate. (b) qPCR analysis of IRF9 mRNA levels after treatment with ribavirin (100 μg/mL for 24 h), B18R (30 ηg/mL for 30h) and IFN-α (100 U/mL for 24 h). Data is obtained from experiments performed in triplicate. Supplementary Figure 7: Induction of ISGs by ribavirin in hepatic stellate cells. (a) Microarray analysis demonstrating gene induction of several ISGs after treatment of activated hepatic stellate cells with ribavirin for 24 hours at a dose of 100 μg/mL. Primary rat hepatic stellate cells were isolated and activated as described in Material and Methods. (b) qPCR analysis of IRF7 mRNA levels from HSCs treated with ribavirin (100 μg/mL) and Interferon-α (100 U/mL) for 24 hours. Microarray analysis was performed on RNA from obtained from the combination of samples from three separate experiments. Supplementary Figure 8: Treatment with ribavirin does not cause increased cell death. Huh7.5.1 cells were treated with ribavirin and etoposide at the indicated doses for 24 hours, and then subjected to cell toxicity assay as described in Materials and Methods. Data is obtained from experiments performed in triplicate.

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