Figure 5

BAY11 inhibits nuclear translocation of IRF7 and NF-κB phosphorylation in pDCs upon TLR-mediated activation. Human pDCs were pretreated for 15 minutes with BAY11 (10^-9 to 10^-7 M) or vehicle, followed by addition of CpG 2216 and cultured for three hours. (A, B) Freshly isolated or activated pDCs were visualized by immunofluorescence with IRF7 antibody (Cy5, red) and nuclei staining with DAPI (blue). Similar results were observed in four independent donors and the representative cells are shown in (A). Cells without nuclear IRF7 expression were counted on the slide from four independent donors (B). Cells were regarded as negative when the expression level of IRF7 in the cytoplasm was higher and distinguishable from that in the nucleus, shown by DAPI staining. Ratio of nuclear IRF7-negative cells was analyzed by 50 cells in each donor. Statistical significance was determined using Mann-Whitney test (**P < 0.01). (C) Staining with anti-p-NF-κB p65 mAb (shaded) and isotype-matched control (solid line) for freshly isolated pDCs or activated pDCs are shown. Similar results were observed in three independent donors and the results of a representative experiment are shown.