Figure 3.

IL-27 differentially affects NF-κB activity in tolerized THP-1 cells relative to PMA–THP-1 cells. THP-1 cells (A) and PMA–THP-1 (B) cells were stimulated with LPS (10 ng/ml), IL-27 (100 ng/ml), or LPS + IL-27 simultaneously for 24 h. Cells were washed and challenged with LPS (100 ng/ml) for 2 h. Cells were stained with anti-NF-κB p65 (red) and NucRed DNA stain (blue). The relative brightness of NF-κB p65 was measured in identically sized regions of the cell nucleus and cytoplasm. The images are representative of three independent experiments. DIC images are shown on the left of the panels. Merged images display overlays of NucRed and p65. A scale bar = 30 μm is displayed at the bottom left for each cell type. The nuclear/cytoplasmic ratios were calculated for each condition (bottom). Graphs present the mean ± S.E. of 18 cells/condition. Three representative images, including those presented here in A and B, also are depicted in Fig. S2, where they are denoted with an asterisk. THP-1 XBlue cells (C) and PMA–THP-1 XBlue cells (D) were stimulated with LPS (10 ng/ml), IL-27 (100 ng/ml), or LPS + IL-27 simultaneously for 24 h (1°). Cells were washed and challenged with LPS (100 ng/ml) for 24 h (2°) to allow for NF-κB–induced SEAP production and secretion. SEAP production was quantified using a colormetric QUANTI-Blue^TM assay. Mean ± S.E. absorbance values are displayed. Data are representative of six independent experiments.