Fig. 4

Zebrafish and human LGP2s play a negative role in regulating IFN response by high concentrations of poly(I:C) in mammalian cells (A–C) DrLGP2 and HsLGP2 downregulated fish IFN and human IFNβ promoter activation by poly(I:C) at a high concentration of 2 μg/ml in mammalian cells. HEK293T cells seeded in 24-wells plates were co-transfected with DrIFNφ1pro-luc or CaIFNpro-luc (A, B), or HsIFNβpro-luc (200 ng each) (C), together with DrLGP2 or HsLGP2 at increasing doses (0, 10, 50, 100, 200 ng). 24 h later, cells were transfected with 2 μg/ml poly(I:C) for another 24 h, followed by luciferase assays. P values were calculated using ANOVA. **P < 0.01. (D) RNA pull-down assays verified the binding of poly(I:C) to DrLGP2 and HsLGP2 in mammalian cells. HEK293T cells seeded in 10 cm dishes were transfected with DrLGP2-HA, HsLGP2-HA or GFP-HA as control. 24 h later, cells were lysed. One-tenth of cell lysates were taken as input, the remaining was incubated with 100 ng biotinylated poly(I:C), followed by western blots with anti-HA antibody. (E–I) Titration of poly(I:C) revealed a negative regulation of zebrafish and human LGP2s on IFN response in mammalian cells. HEK293T cells seeded in 24-wells plates were co-transfected with HsIFNβpro-luc (E, H, I), or with DrIFNφ1pro-luc or CaIFNpro-luc (F, G), together with DrLGP2 (E, F, H) or HsLGP2 (G, I) (200 ng each). Renilla vector (pRL-TK, 0.2 ng) was transfected as internal control. 24 h later, cells were transfected with poly(I:C) at increasing doses for another 24 h, followed by luciferase assays. P values were calculated using Student’s t-test. **P < 0.01.