PLIC-1 Inhibits TLR3 Activation Figure 1. Overexpression of PLIC-1 inhibited TLR3 signaling. A. TLR4/MD2 stable HEK cells were transfected with a NF-kB driven luciferase reporter plasmid and increased amount of HA-PLIC-1. 24 hours post-transfection, cells were stimulated with LPS (100 ng/ml) and incubated for additional 24 hours prior to luciferase reporter assay. B. 293T cells were transfected with pISRE-Luc (100 ng), CD4-TLR4 (50 ng), and increased amount of HA-PLIC-1 for luciferase reporter assay. C. 293T cells were transfected with Trif, kB-Luc, and increased amount of HA-PLIC-1 plasmids. A total of 2 mg DNA was transfected in each well in a 12-well plate. 48 hours following transfection, luciferase activity was determined. Data was normalized against renilla luciferase which was transfected as an internal control. Experiments in D were carried out similarly as in C except that p125-Luc, plasmid was used. E. p125-Luc reporter was transfected along with Trif and YFP-PLIC-1 plasmids. F. Experiments were carried out similarly as in D except that 0.3 mg IRF3 expression plasmid was added to stimulate p125-luc reporter. G. HEK cells stably expressing TLR7 were transfected with indicated constructs and stimulated with TLR7 agonist, imiquimod (10 mg/mL) for 24 hours followed by luciferase reporter assay. H. 293T cells were transfected with MAVS and increasing amount of PLIC-1 for reporter assays. I. 2.56105 J774A.1 cells were transfected with indicated plasmids. Poly I:C(40 mg/ml) was added to cells for additional period of 24 hours prior to measuring luciferase activity. Data were normalized by using renilla luciferase as an internal control. The error bars in above experiments represent the standard deviation accumulated from at least three experiments. doi:10.1371/journal.pone.0021153.g001 the lung epithelial cell line A549 which supports infection of a NDV virus carrying the green fluorescent protein (GFP) in its genome. Because NDV is highly sensitive to type I interferon treatment, such a system allows rapid and sensitive monitoring of bioactivity of type I interferon. Shown in Fig. 3, polyI:C stimulation of A549 cells modestly reduced the NDV-GFP infection, while the inhibition was significantly enhanced in PLIC-1 knockdown cells. When the bioactivity of type I interferon PLoS ONE | www.plosone.org 3 June 2011 | Volume 6 | Issue 6 | e21153