Figure 4.

NF-κB activity is reduced in miR-718-overexpressing macrophages stimulated with LPS. A, 293T cells were transiently transfected with TLR4, NF-κB-Luc (firefly), control (Renilla) Luc, the miR-718 construct, or Control miR constructs and then stimulated with various concentrations of LPS for 2 h. After 48 h, 293T cells were lysed, and the normalized firefly luciferase activity (firefly luciferase activity/Renilla luciferase activity) was calculated. RLU, relative light units. B, whole cell extracts from Con miR and miR-718 macrophages stimulated with 100 ng/ml LPS for 2 h were analyzed for IκBα, P65, and β-actin protein expression by immunoblotting. Data are representative of three independent experiments. Med, medium. C, Con miR and miR-718 macrophages stimulated with 100 ng/ml LPS for 2 h were fixed, permeabilized, and stained with Alexa Fluor 647 anti-p65 NF-κB subunit antibody and DAPI (nucleus) and subjected to confocal microscopy. Scale bars = 20 μm. Images are representative of at least 10 fields of view and three independent experiments. D, the presence of nuclear P65 was quantified in 10–15 fields by confocal microscopy. Mean ± S.D. of triplicate determinations are shown. *, p < 0.05; **, p < 0.005.