Effects of RNAi-mediated knockdown of DYRK2 on SeV-induced signaling and IRF3 activation.

Abstract

<p>(A) Effects of DYRK2 RNAi on the expression of transfected and endogenous DYRK2. In the upper panel, 293 cells (2×10<sup>5</sup>) were transfected with expression plasmids for Flag-DYRK2 and HA-NEK6 (0.1 μg each) and the indicated RNAi plasmids (1 μg). Twenty-four hours after transfection, the cell lysates were analyzed by immunoblotting with the indicated antibodies. In the lower panel, the 293 cells (2×10<sup>5</sup>) were transfected with the control or indicated DYRK2 RNAi plasmids (1 μg each) for 36 h. The cell lysates were analyzed by immunoblotting with the indicated antibodies. (B) Effects of DYRK2 RNAi on the SeV-induced activations of the ISRE, NF-κB and the IFN-β promoter. The 293 cells (1×10<sup>5</sup>) were transfected with the ISRE, NF-κB or IFN-β promoter reporters (0.05 μg) and the indicated RNAi plasmids (0.5 μg each) for 36 h and then infected or not infected with SeV for 10 h before the luciferase assays were performed. The graphical data are presented as the means ± the SDs (n = 3). (C) Knockdown of DYRK2 promoted SeV-induced IRF3 dimerization. The 293 cells (2×10<sup>5</sup>) were transfected with control or DYRK2 RNAi (#2) plasmids (1 μg). Thirty-six hours after transfection, the cells were infected with or without SeV for 0, 4, 8, or 12 h. The cell lysates were separated by native (top) or SDS (bottom) PAGE, and the blots were analyzed using the indicated antibodies. (D) Effects of DYRK2 RNAi on the SeV-induced endogenous gene transcriptions of IFNB1 and RANTES. The 293 cells (2×10<sup>5</sup>) were transfected with the indicated RNAi plasmids (1 μg each) for 36 h and then infected or not infected with SeV for 10 h before reverse transcription PCR was performed. (E) Effects of DYRK2 RNAi on SeV-, VSV- and HSV-1-induced transcriptions of IFNB1 and RANTES in THP-1 cells. RNAi-transduced stable THP-1 cells (2×10<sup>5</sup>) were infected or not infected with SeV/VSV/HSV-1 for 8 h before qPCR was performed. (F) Effects of DYRK2 RNAi on SeV- and HSV-1-induced secretion of IFN-β. RNAi-transduced stable THP-1 cells (1×10<sup>5</sup>) were infected with SeV or HSV-1 for 12 h. The culture medium was collected for quantitation of the indicated cytokines by ELISA. (G) Effects of DYRK2 RNAi on virus replication. RNAi-transduced stable THP-1 cells (1×10<sup>5</sup>) were mock-transfected or transfected with poly(I:C) (1μg) for 16 h and then infected with VSV or HSV-1 (MOI = 0.1). The supernatants were harvested 24 h after infection for standard plaque assays.</p

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