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. 2013 Mar;87(6):3039–3052. doi: 10.1128/JVI.03176-12

Fig 4.

Fig 4

NS1 downregulates RhoA protein expression by inhibiting NF-κB activation. (A) A549 cells were mock infected (mock) or infected with virus and, after 24 h postinfection, the cells were collected and analyzed by Western blotting with antibodies as indicated. The data represent mean fold changes ± the SD of three independent experiments (*, P < 0.05 relative to the control group). (B) The expression level of RhoA mRNAs in transfected A549 cells was assessed using real-time PCR after transfection of the relevant plasmids for 24 h. GAPDH mRNA values were used for normalization. The relative mRNA expression of pcDNA3.0 set as 1. The data represent the mean fold changes ± the SD of three independent experiments (*, P < 0.05 relative to the control group). The expression of Flag-NS1 was monitored by Western blotting with an anti-Flag antibody. (C) Cells were transfected with plasmids or treated with reagents as indicated. pRL-TK-RhoA promoter activity was measured using a dual-luciferase reporter assay system (Promega) after 24 h. hRluc luciferase reporter vector pGL3.0 (Promega) was used as an internal control for transfection efficiency. (D) RhoA promoter and NF-κB binding site deletion sequences were both cloned upstream of luciferase. The candidate NF-κB binding site sequence is shown at the bottom. The cells were harvested at 24 h posttransfection and assayed for luciferase activity. The data represent the mean fold changes ± the SD of three independent experiments (*, P < 0.05 relative to the control group). (E) The A549 and Vero cells were cotransfected with pNF-κB-Luc, pRL-tk (internal control), and the plasmid or reagent as indicated. At 30 h posttransfection, the cells were treated with TNF-α (10 ng/ml) for 4 h, and the luciferase activity was assayed. The data represent the mean fold changes ± the SD of three independent experiments (*, P < 0.05 relative to the control group). (F) A549 cells and Vero cells were cotransfected with pNF-κB-Luc or pRL-tk (internal control) and infected with virus as indicated. Luciferase activity was assayed 24 h later. The data are shown as means ± the SD of three independent experiments (*, P < 0.05 relative to the control group).