NOD2 expression increased in osteoarthritis (OA) cartilage and 29-kDa FN-f-treated chondrocytes. (A) Relative expression of NOD2 in human normal and OA cartilage. Relative expression of NOD2 in normal and OA cartilage was measured using SYBR Green-based real-time polymerase chain reaction (qPCR). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an endogenous control. *P < 0.05 vs. normal cartilage. Data are presented as the mean ± standard deviation (SD) of data from duplicate experiments using cartilages from different donors (normal cartilage, n = 7 and OA cartilage, n= 25). (B) IHC staining for NOD2 in normal and OA cartilage. NOD2 expression was determined by IHC using an antibody against NOD2. Sections were counterstained with methyl green. Data are representative of results from three normal and OA cartilages. Scale bars = 100 μm for 100× and 200× magnifications. The arrows indicate NOD2-positive chondrocytes. (C) 29-kDa FN-f and inflammatory cytokines, including (D) IL-1β and (E) TNF-α, positively increased NOD1 and NOD2 expression. Chondrocytes were exposed to 29-kDa FN-f, IL-1β, or TNF-α for 6, 24, and 48 h and mRNA levels of NOD1 and NOD2 were measured by qPCR. Data are presented as the mean ± SD of duplicate data from more than five different donors. *P < 0.05, ***P < 0.005, and ****P < 0.001 vs. untreated control. ns, not significant.