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. 2010 Dec 8;5(12):e14258. doi: 10.1371/journal.pone.0014258

Figure 2. DDX3 is a positive regulator of IPS-1-mediated IFN promoter activation.

Figure 2

(A) IFN-β induction by polyU/UC is augmented by DDX3. IPS-1 (100 ng), DDX3 (100 ng) and p125luc reporter (100 ng) plasmids were transfected into HEK293 cells in 24-well plates with or without the HCV 3′ UTR poly U/UC region (PU/UC) RNA (0, 25 or 50 ng/well), synthesized in vitro by T7 RNA polymerase. HCV RNA-enhancing activation of IFN-beta promoter was assessed by reporter assay in the presence or absence of the DDX3-IPS-1 complex. (B) RIG-I is essential for the DDX3/IPS-1-mediated IFN-promoter activation. MEF from wild-type and RIG-I −/− mice were transfected with plasmids of IPS-1, DDX3 and p125luc as in panel A, and stimulated with polyU/UC (0, 25 or 50 ng/well). Reporter activity was determined as in panel A. (C) Knockdown of DDX3. Negative control or DDX3 targeting siRNA (20 pmol), DDX3 si-1, was transfected into HEK293 cells, and after 48 hrs, expression of endogenous DDX3 mRNA was examined by real-time RT-PCR. DDX3 si-1-mediated down-regulation of the DDX3 protein was also confirmed by Western blotting (data not shown). (D) DDX3 enhances RIG-I-mediated IFN-beta promoter activation induced by polyU/UC. DDX3 si-1 or control siRNA was transfected into HEK293 cells with reporter plasmids (100 ng). After 48 hrs, cells were stimulated with polyU/UC (5∼50 ng/ml) with lipofectamin 2000 reagent for 6 hrs, and activation of the reporter p125luc was measured. The results are representative of at least two independent experiments, each performed in triplicate.