FIGURE 7. S100A9 is essential for TLR3 targeting to polyIC positive intracellular compartment.

(A) Co-immunofluorescence analysis was performed by treating primary bone marrow derived macrophages (BMDM) isolated from wild-type (WT) and S100A9 knockout (KO) mice with biotinylated-polyIC (polyIC). Following treatment, cells were fixed and labeled with Avidin-FITC (green) and TLR3 (Texas red - red) antibody. The images of polyIC (8h treatment) treated WT cells represent 69% of cells (i.e. 69 cells out of 100 cells) with polyIC and TLR3 co-localization. We failed to detect polyIC and TLR3 co-localization in S100A9 KO cells. (B) BMDM isolated from WT and S100A9 KO mice were treated with biotinylated-polyIC (polyIC) for co-IFA analysis. Following polyIC treatment, cells were fixed and labeled with Avidin-FITC (green) and antibody specific for late endosome marker Rab9 (Texas red - red). The images of polyIC treated WT and S100A9 KO cells represent 67% and 59% of cells (i.e. 67 cells or 59 cells out of 100 cells) with polyIC and Rab9 co-localization, respectively. Merged images in (A) and (B) are represented as yellow. The images are representative of thirty viewing fields from two independent experiments with similar results. The white bar represents 10 μm. White arrow heads in merged images represents co-localization. UT; untreated cells.