Curcumin inhibits IL-1β activity, enabling co-culture induced chondrogenesis in MSCs. A: Fourteen days high-density culture. Untreated MSC cultures became apoptotic (a). In primary chondrocyte cultures (b), co-cultures (c), co-cultures treated with curcumin (e) or co-cultures pre-stimulated four hours with curcumin (f), prominent chondrogenesis was observed. Stimulation of the co-culture with IL-1β alone resulted in degeneration of the cell culture (d). In contrast, a four-hour pre-stimulation of the co-culture with curcumin followed by IL-1β incubation (g) or a four-hour pre-stimulation of the co-culture with curcumin followed by IL-1β and curcumin incubation (h) inhibited the adverse effects of IL-1β on the chondrogenic potential of the co-culture and prominent chondrogenesis was observed. Magnification, 6,000×; bar, 1 μm; C, chondrocytes, F, fibroblast-like cells; M, ECM. B-D: Immunoblots of whole cell lysates were probed with antibodies against CSPGs, collagen type II, β1-integrin, Shc, activated-ERK1/2, Sox-9, activated-caspase-3 and COX-2. In co-cultures pre-stimulated for four hours with curcumin followed either by incubation with IL-1β alone or incubation with IL-1β and curcumin, prominent production of chondrogenic matrix and adhesion molecules (B), activation of the chondrogenic signalling pathway (C) and down-regulation of apoptotic and inflammatory markers (D) was observed. Each experiment was performed in triplicate. Expression of the housekeeping gene β-actin was not affected.