Figure 5

Cathepsin S/B expression and activity is enhanced under cytokine stimulation. (A) Fold-increase in cathepsin S and B (denoted as CATS/B) expression in cytokine (that is, TNFα and IL1β) stimulation of monolayer cultures (2D) and chondrocyte cultures in alginate microbeads (3D), normalized to GAPDH and untreated cells. For treated 3D cultures cytokines were added to the crosslinked alginate suspension containing chondrocytes. Unt indicates untreated controls. (B) Human chondrocytes were isolated from non-OA/normal and osteoarthritis (OA)-diagnosed donors, mRNA was isolated from samples and analyzed for CATS expression. (C) Protein extracts from human OA-derived chondrocytes (passage 0) that were treated or untreated with cytokines were immunoblotted for cathepsin S and B (D) and cystatin C. Band quantification plot beneath each blot in (C) and (D). (E) Left panel shows immunohistochemistry (IHC) of IC/DC sections stained for cathepsin S, middle panel for cathepsin B and right panel for cystatin C (n = 3). Statistical analysis was by non-parametric Mann-Whitney analysis, assuming P <0.05 to be statistically significant.