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. 2011 Sep;31(18):3802–3819. doi: 10.1128/MCB.01368-10

Fig. 9.

Fig. 9.

DDX60 promotes RIG-I- or MDA5-mediated signaling. (A to C) Activation of the IFN-β promoter was examined using a reporter gene assay and p125luc plasmid. Vectors expressing RIG-I (A), MDA5 (B), DDX6 (C), and the wild type (WT) or DDX60-K791A (C) were transfected into HEK293 cells together with the reporter plasmid and Renilla luciferase plasmid (internal control). After 24 h, the cells were left unstimulated or stimulated with poly(I·C) by the use of DEAE-dextran for 4 h. Cell lysates were prepared, and luciferase activity was measured. (D) Control or DDX60 knockdown HEK293 cells were transfected with the p125luc reporter, Renilla luciferase plasmid, and/or in vitro-synthesized VSV dsRNA. After 24 h, cell lysates were prepared and luciferase activity was measured. (E and F) siRNA for DDX60 or control siRNA was transfected into HEK293 cells. The cells were left unstimulated or stimulated with poly(I:C), and expression of IFN-β and DDX60 mRNA was measured by RT-qPCR. Expression values were normalized using GAPDH. (G) siRNA for DDX60 or the control was transfected into HEK293 cells together with DDX60-expressing vector. The DDX60 protein was observed by Western blotting. (H) Vectors expressing TICAM-1 and/or DDX60 were transfected into HEK293 cells together with the p125luc reporter and Renilla luciferase plasmids. After 24 h, the cell lysates were prepared and luciferase activities were measured. (I) Vectors expressing TLR3 and/or DDX60 were transfected into HEK293 cells together with the p125luc reporter and Renilla luciferase plasmids. After 24 h, the cells were left unstimulated or stimulated with poly(I:C) for 4 h, the cell lysates were prepared, and luciferase activity was measured. (J and K) HeLa cells expressing shRNA for DDX60 (J) or EXOSC4 (K) were stimulated with 50 μg/ml of poly(I·C) (no transfection) (J) or dsRNA (transfection) (K). RT-qPCR was performed to measure IFN-β mRNA expression. (L) HeLa cells expressing shRNA for GFP, EXOSC4, or EXOSC5 were infected with VSV at an MOI of 1. Levels of induction of IFN-β mRNA were calculated as described for panel J. (M and N) Empty or IPS-1-expressing vector (M) and RIG-I CARD-, MDA5-, or TBK1-expressing vector (N) were transfected into control or DDX60 knockdown HEK293 cells together with p125luc reporter and Renilla luciferase plasmids. After 24 h, cell lysates were prepared and luciferase activity was measured. (O) shRNA for DDX60 did not inhibit the signaling from TLR3 (H to J). Although DDX60 promotes RLR-dependent signaling (A to E), shRNA for DDX60 did not reduce the signaling induced by RIG-I CARD, MDA5, IPS-1, or TBK1 overexpression (M and N). These data suggest that shRNA suppresses signaling upstream of RIG-I and MDA5.