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. 2012 May 16;7(5):e37398. doi: 10.1371/journal.pone.0037398

Figure 5. Validation of synergy between ZAP and the top three ISGs in a knockdown system.

Figure 5

A) Triplicate wells of Huh-7 cells were transfected with irrelevant siRNA, ZAP-specific siRNA, ISG-specific siRNA that targets IRF2, RIG-I or IL28RA, or siRNAs that target both ZAP and an ISG. ISG-specific siRNA was added to cells again on the second day after seeding. Forty-eight h after initial siRNA transfection, cells were infected with Toto1101/Luc (moi = 5). Viral replication was determined by firefly luciferase activity 4 h after infection. Huh-7 cells that were not transfected with siRNA were included as a negative control. Means and standard deviations of triplicate samples are shown. Asterisks indicate mean values statistically different between two siRNA treatments (unpaired t test, *, P<0.05; **, P<0.01; ***, P<0.001). B) Forty-eight h after initial siRNA transfection, total RNA was extracted from the cells and used to generate cDNA. RNA levels of IRF2, RIG-I, IL28RA and RPS11 were measured by real-time PCR. The ISG mRNA levels were normalized with that of RPS11, and the ISG mRNA levels in irrelevant siRNA-transfected cells were set as 1. Data are means +/− SD of one experiment in triplicate.