Fig. 6

Colocalization of rhodamine-labeled recombinant human proteoglycan-4 (rhPRG4) (red) and isotype control (IC), CD44 (probed using anti-CD44), toll-like receptor 2 (TLR2) (probed using anti-TLR2) or toll-like receptor 4 (TLR4) (probed using anti-TLR4) in peritoneal Prg4^−/− murine macrophages. Cells were incubated with rhodamine-rhPRG4 for 2 h followed by cell fixation and permeabilization. Following receptor probing, cells were incubated with Alexa Fluor 488 conjugated secondary antibody (green) and counterstained with DAPI (blue). Arrows point to co-localization of rhPRG4 with respective receptors. Quantitative colocalization analysis was performed using Pearson’s Correlation Coefficient and a cutoff of r² > 0.5 was used to indicate positive colocalization. The percentage of cells with positive colocalization was determined and at least 100 cells were examined for each treatment condition. Data represent the mean ± S.D. of three independent experiments. Median colocalization images are presented. *p < 0.001; **p < 0.01; ***p < 0.05. Scale = 20μm. a Representative image of rhodamine-rhPRG4 treated Prg4^−/− macrophages and probed with IC antibody. b Representative image of rhodamine-rhPRG4 treated Prg4^−/− macrophages and probed with anti-CD44 antibody. c Representative image of rhodamine-rhPRG4 treated Prg4^−/− macrophages and probed with anti-TLR2 antibody. d Representative image of rhodamine-rhPRG4 treated Prg4^−/− macrophages and probed with anti-TLR4 antibody. e Colocalization of rhPRG4 and CD44 was higher compared to rhPRG4 and TLR2 colocalization and rhPRG4 and TLR4 colocalization