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. 2007 Apr 25;104(18):7500–7505. doi: 10.1073/pnas.0611551104

Fig. 3.

Fig. 3.

Suppression of RIG-I function by ubiquitination. (a) The RNF125 suppressed the IFNβ-driven luciferase activity, activated by RIG-I (at the left). The 293FT cells were transfected with plasmids encoding RIG-I (50 ng) and IFNβ-luc (50 ng) with varying amounts of a plasmid encoding RNF125 (10, 50, and 100 ng). Twenty-four hours after transfection, cells were treated with polyIC (blue) or infected with Sendai virus (pink). Cells were harvested 12 h after the treatment; luciferase activity in the lysates was then measured. The RNF125 suppressed the production of IFNβ mRNA. (b) The 293FT cells transiently expressing IFNβ-luc, RIG-I, and RNF125WT or Δ76 were analyzed for luciferase activity 36 h after transfection. (c) Effect of siRNF125–3, a small inhibitory RNA specific for RNF125 mRNA, on IFNβ-luc activity and IFNβ level. The 293FT cells were transfected with plasmids encoding IFNβ-luc and RIG-I and treated with control siRNA or siRNF125–3. An aliquot of cells was then infected with Sendai virus. Twenty-four hours after infection, luciferase activity (Left) and IFNβ mRNA levels, assessed by RT-PCR (Right), were measured. (d) Effect of mouse RNF125 on endogenous RIG-I signaling in primary MEFs. The MEFs were transfected with plasmids encoding mouse RNF125 (100 ng in lanes 4, 5, 6, and 8 or 200 ng in lane 9) and IFNβ-luc (50 ng) in combination as indicated. Twelve hours after transfection, cells were infected with Sendai virus at the indicated multiplicity of infection (MOI) or mock infected. Luciferase activity in cell lysates prepared 24 h after treatment was measured. White box, mock infected; pink box, Sendai virus infected with MOI 1; black box, Sendai virus with MOI of 10. (e) The MEFs were transfected with plasmids encoding IFNβ-luc and treated with control siRNA or siRNF125–3 (final concentration of 8 nM). Cells were then infected with Sendai virus at MOI of 1. Twenty-four hours after infection, luciferase activity was measured. (f) The level of IFNβ in culture medium in cells was decreased in an RNF125 dose-dependent manner. Cells transfected with different amounts of plasmids encoding RNF125 were treated with 0 (green), 2 (pink), and 20 (blue) μg/ml of poly I:C (Upper) and with 0 (green), 1 (pink), and 10 MOI (blue) of Sendai virus (Upper). Twelve hours after the treatment, IFNβ in the culture medium was measured by ELISA system. (g) The level of IFNβ was increased in MEFs treated with siRNA for RNF125-specific mRNA. Cells treated with control siRNA (black bars) or siRNA125–3 (pink bars) were treated with different doses of poly I:C or Sendai virus infection and were analyzed for IFNβ in culture medium.