TSG-6 interferes with LPS-induced activation of NF-κB signaling. BV2 cells were stimulated with LPS in the presence and absence of TSG-6 (10 ng/ml). Cells were immunostained with a primary antibody against NF-κB p65, followed by an Alexa Fluor 594-conjugated secondary antibody. Actin filaments (green) and cell nuclei (blue) were visualized with FITC-labeled phalloidin and DAPI separately. Cell images were obtained using a confocal microscopy. (A) Typical micrographs of immunocytochemistry are shown for cytoplasmic and nuclear distribution on NF-κB p65. Scale bars, 30 μm. (B) The percentages of cells with NF-κB p65 localized to the nucleus were determined by analysis of at least 100 cells per slide. (C) Nuclear extracts were prepared and processed for chemiluminescence-based NF-κB EMSA experiments. Cells were stimulated with 100 ng/ml LPS with or without TSG-6(10 ng/ml) for the indicated periods. Nuclear extracts were incubated with a biotin-labeled NF-κB-specific oligonucleotide and further probed with streptavidin-HRP. The arrow indicates shifted DNA probe for NF-κB and free probe respectively. (D) BV2 cells were co-transfected with pNF-κB-luciferase reporter plasmid and pRL-TK control plasmid and then treated with or without LPS (100 ng/ml) in appearance or absence of rmTSG-6 (10 ng/ml) for 6 hours. NF-κB activities were measured by luciferase assay, normalized to luciferase activities of pRL-TK, and quantified as fold changes over the control (unstimulated BV2 cells). Values are mean ± SD. n = 3; **P <0.01; *P <0.05; significantly different from LPS-treated cells. Abbreviations: LPS, lipopolysaccharide; NF-κB, nuclear factor (NF)-κB.