FIG 4.

RIG-I and DDX60 regulated by NEAT1 facilitates HTNV-induced IFN-β production. (A) HUVECs transfected with NC or si-NEAT1-2 were mock infected or infected with HTNV at an MOI of 1, and expression levels of TLR1, TLR2, TLR3, TLR4, RIG-I, DDX60, and MDA5 were measured by qRT-PCR at 48 hpi. Values are means ± SD (n = 3; *, P < 0.01; **, P < 0.001; Student's t test). (B and C) HUVECs with increasing amounts of si-NEAT1-2 (B) or pCMV-NEAT1-2 (C) were infected with HTNV at an MOI of 1, and the RIG-I and DDX60 production levels were measured by Western blotting at selected time points. (D) HUVECs infected with HTNV at an MOI of 1 were subjected to visualization of RIG-I (red) and DDX60 (green) by immunostaining at 48 hpi; counterstaining was conducted with DAPI (blue). (E and F) HEK293 cells were transfected with NC sequences, si-RIG-I-2, si-DDX60-1, or both si-RIG-I-2 and si-DDX60-1. Twenty-four hours after transfection, the cells were infected with HTNV at an MOI of 1. The knockdown efficiencies of RIG-I or DDX60 (E, upper panels), as well as HTNV NP expression (E, lower panels), were assessed at 48 hpi by Western blotting. qRT-PCR was performed to detect the expression of IFN-β. (F) Values are means ± SD (n = 5; *, P < 0.01; **, P < 0.001; one-way ANOVA). (G) A luciferase gene harboring the IFN-β promoter was transfected with si-RIG-I-2 and/or si-DDX60-1 in HEK293 cells, and after 24 h, the HEK293 cells were infected with HTNV at an MOI of 5. At 48 hpi, the relative luciferase activity for IFN-β was compared with that in cells that received a mock transfection, as determined by the luminescence intensity. Values are means ± SD (n = 5; *, P < 0.01; **, P < 0.001; one-way ANOVA). (H and I) HEK293 cells were transfected with vector plasmids, Flag-RIG-I, pUNO-DDX60, or both Flag-RIG-I and pUNO-DDX60. Twenty-four hours after transfection, the cells were infected with HTNV at an MOI of 1. The overexpression efficiencies of RIG-I or DDX60 (H, upper panels), as well as HTNV NP expression (H, bottom panels), were assessed at 48 hpi by Western blotting. Additionally, qRT-PCR was performed to detect the expression of IFN-β (I) in HEK293 cells. Values are means ± SD (n = 5; *, P < 0.01; **, P < 0.001; one-way ANOVA). (J) A luciferase gene harboring the IFN-β promoter was transfected with Flag-RIG-I and/or pUNO-DDX60 in HEK293 cells. Twenty-four hours later, the cells were infected with HTNV at an MOI of 5. At 48 hpi, the relative luciferase activity for IFN-β was compared with values for mock-transfected cells, as determined by the luminescence intensity. Values are means ± SD (n = 5; *, P < 0.01; **, P < 0.001; one-way ANOVA). The experiments were performed independently at least three times with similar results.