Cathepsin activity is increased in degraded cartilage from osteoarthritis (OA) patients. Intact and degraded cartilage (IC and DC) derived from end-stage OA patients were analyzed with GB123 for (A) cathepsin activity in freshly isolated chondrocyte lysates and (B) degraded cartilage media, which was obtained following a collagenase digestion procedure (24 h, 37°C). Samples were treated with GB123 and subjected to SDS PAGE and fluorescence visualization as in Figure 1 (n = 4). The heavy chain of active Cathepsin L was detected between 20 to 25 kDa (A). (C) Recombinant cathepsin B (right) and cathepsin S (left) were treated for 24 h with collagenase digestion media (0.02%), labeled with GB123 and analyzed as in Figure 1 (n = 3). (D) Enzymes were analyzed by SDS-PAGE and immunoblotted with cathepsin B or S antibodies (n = 3). (E,
F) Cryosections of IC/DC (5 μm-thick) stained with 4′: 6-diamidino-2-phenylindole (DAPI) (blue fluorescence for nuclei), GB123 (red fluorescence for cathepsin activity), with or without pre-incubation with the cathepsin inhibitor GB111-NH2 (n = 4). Increased cathepsin activity in DC versus IC regions is clearly observed. Images taken with 60-times magnification (E) and with 10-times magnification (n = 5) (F). Plots of band intensity (panels A and B; imageJ software) show SD surrounding an average data point. Statistical analysis was by non-parametric Mann-Whitney analysis, assuming P <0.05 to be statistically significant.