ω-3 PUFAs antagonize NF-κB signaling in macrophages.

<p>(A) ω-3 PUFAs suppress TLR4/NF-κB signaling in transfected 293T cells. 293T cells were transfected with TLR4/MD-2 expression vectors, and NF-κB luciferase reporter constructs. Transfected cells were pre-treated with ω-3 PUFA mixtures EPA/DHA (50 µM each) for 24 hours and then stimulated with LPS (100 ng/ml) in the presence or absence of ω-3 PUFA for additional 24 hours. NF-κB luciferase activity was measured using a Dual-Luciferase Reporter Assay. A.U.: Arbitrary Units. (B) ω-3 PUFAs suppress the NF-κB signaling in macrophages. Raw264.7 macrophages were transfected with NF-κB luciferase reporter constructs alone. The treatment is the same as described in (A). (C) ω-3 PUFAs act on the downstream signal(s) of TLR4 to inhibit NF-κB. Raw264.7 macrophages were transfected with CA-MyD88 expression vectors and NF-κB luciferase reporter constructs. (D) ω-3 PUFAs blocks NF-κB DNA binding. EMSA was conducted to examine the NF-κB DNA binding and was conducted as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045990#s4" target="_blank">Materials and Methods</a>. (E) ω-3 PUFAs blocks the NF-κB subunit p65 binding to the IL-6 promoter. ChIP assays were conducted to examine p65 DNA binding and were conducted as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045990#s4" target="_blank">Materials and Methods</a>. SYBR Green quantitative PCR was used to quantitate the immunoprecipitated DNA. For (A)–(C), all data are expressed as mean ± SE, n = 6. Groups labeled with the same superscripts are not statistically different from each other. Groups labeled with different superscripts are statistically different from each other.