Figure 5

K-80003 reduced NF-κB activity in rat primary chondrocytes and joints. Rat primary chondrocytes were transfected with RXRα SiRNA (0.5 μM), or ERα SiRNA (0.5 μM), or control siRNA (0.5 μM) with Lipofectamine 3000 for 24 h, following by incubation with vehicle, or K-80003 (5 μM, 10 μM) or 17β-estradiol (10 nM) for 30 min. Cells were then treated with IL-1β (10 ng/mL) for 48 h. (A) Representative western-blot bands and quantification of of p-p65 and p-IκBα abundances in chondrocytes. (B) Confocal fluorescence imaging of p65 in chondrocytes. Green, p65; blue, nuclei; yellow arrows indicate nuclear translocation of p65. (C) Quantification of p65 nuclear translocation. Cells displaying p65 in nuclear localization are counted and expressed as a percentage of the total number of chondrocytes. (D) Immunofluorescence analyses and quantification of p-p65 in the tibia medial compartment of animals. ***P < 0.001; n = 8.