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. 2009 Dec 22;107(2):815–820. doi: 10.1073/pnas.0908967107

Fig. 3.

Fig. 3.

Knockdown of WDR5 inhibits SeV-induced activation of IRF3, NF-κB and the IFN-β promoter (A) Effects of WDR5-RNAi plasmids on the expression of WDR5. 293 cells (2 × 105) were transfected with expression plasmids for Flag-WDR5 and Flag-V59 (0.1 μg each), and the control or indicated WDR5 RNAi plasmids (1 μg). Twenty-four hours after transfection, cell lysates were analyzed by immunoblot with anti-Flag. The WDR5 bands from three independent experiments were quantitated using the Bio-Rad Quantity One Program and normalized by levels of the control protein V-59. The average levels of WDR5 from the three experiments are shown at the bottom of the blot. **, P < 0.01, n = 3. (B, D, and F) Effects of WDR5-RNAi plasmids on SeV-induced activation of the IFN-β promoter (B), ISRE (D), and NF-κB (F); 293 cells (2 × 105) were transfected with the indicated RNAi plasmids together with the indicated reporter plasmids. Cells were left uninfected or infected with SeV for 8 h before luciferase assays were performed. Graphs show mean ± SD, n = 3. **, P < 0.01. (C) Effects of WDR5 knockdown on transcription of endogenous IFNB1 gene. 293 cells (2 × 105) were transfected with a control or the indicated WDR5-RNAi plasmids. Twenty-four hours after transfection, cells were infected with SeV for 6 h or left uninfected before RT-PCRs were performed with the indicated primers. (E) Knockdown of WDR5 inhibits SeV-induced IRF3 phosphorylation; 293 cells (2 × 105) were transfected with a control or the indicated WDR5-RNAi plasmids. Twelve hours after transfection, cells were selected with puromycin (1 μg/mL) for 24 h, then infected with SeV for 6 h or left uninfected. Cell lysates were analyzed by immunoblots with the indicated antibodies. (G) WDR5-RNAi does not inhibit IL-1-induced NF-κB activation. 293 cells (2 × 105) were transfected with NF-κB reporter plasmid together with a control or WDR5-RNAi plasmid. Twenty-four hours after transfection, cells were left untreated or treated with IL-1 (20 ng/mL) for 8 h before luciferase assays were performed. Graphs show mean ± SD, n = 3. (H) Knockdown of WDR5 inhibits RIG-I-, RIG-I-CARD, VISA-, MITA- but not TBK1-mediated ISRE activation. 293 cells (2 × 105) were firstly transfected with a WDR5-RNAi or control plasmid (1 μg). Twenty-four hours later, cells were selected with puromycin (1 μg/mL) for 24 h and then retransfected with ISRE reporter and the indicated expression plasmids (0.1 μg each). Luciferase assays were performed 24 h after the second transfection. Graphs show mean ± SD, n = 3. *, P < 0.05; **, P < 0.01.