Differential regulation of proteoglycan 4 metabolism in cartilage by IL-1α, IGF-I, and TGF-β1 T.A. Schmidt, Ph.D., N.S. Gastelum, B.S., E.H. Han, M.S.,

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Differential regulation of proteoglycan 4 metabolism in cartilage by IL-1α, IGF-I, and TGF-β1 T.A. Schmidt, Ph.D., N.S. Gastelum, B.S., E.H. Han, M.S., G.E. Nugent-Derfus, Ph.D., B.L. Schumacher, B.S., R.L. Sah, M.D., Sc.D. Osteoarthritis and Cartilage Volume 16, Issue 1, Pages 90-97 (January 2008) DOI: 10.1016/j.joca.2007.05.009 Copyright © 2007 Osteoarthritis Research Society International Terms and Conditions

Fig. 1 Effect of culture conditions on PRG4 presence and expression by chondrocytes in cartilage explants containing the superficial zone. Disks were cultured in medium supplemented with 25μg/ml ascorbic acid, ±10% FBS, as well as 10ng/ml IL-1α, 300ng/ml IGF-I, or 10ng/ml TGF-β1. PRG4 was probed with mAb 3A4 in disks after 6 days of culture. A non-specific IgG was used for the control. Osteoarthritis and Cartilage 2008 16, 90-97DOI: (10.1016/j.joca.2007.05.009) Copyright © 2007 Osteoarthritis Research Society International Terms and Conditions

Fig. 2 Effect of culture conditions on depth-dependent PRG4 expression by chondrocytes in cartilage explants containing the superficial zone, represented as cells per area. PRG4 was probed with mAb 3A4 in disks after 6 days of culture. Data are mean±s.e.m. (n=6–7). ***=P<0.001. Osteoarthritis and Cartilage 2008 16, 90-97DOI: (10.1016/j.joca.2007.05.009) Copyright © 2007 Osteoarthritis Research Society International Terms and Conditions

Fig. 3 Effect of culture conditions on the amount of PRG4 bound to the surface of cartilage explants containing the superficial zone after 6 days of culture. Data are mean±s.e.m. (n=5). *=P<0.05; +=P<0.05, ++=P<0.01, +++=P<0.001 compared to day 0. Osteoarthritis and Cartilage 2008 16, 90-97DOI: (10.1016/j.joca.2007.05.009) Copyright © 2007 Osteoarthritis Research Society International Terms and Conditions

Fig. 4 Effect of culture conditions on PRG4 secretion by chondrocytes in cartilage explants containing the superficial zone. PRG4 secretion rates are shown averaged over 6 days of culture. Shaded regions indicate levels at or below assay sensitivity. Data are mean±s.e.m. (n=8). *=P<0.05. Osteoarthritis and Cartilage 2008 16, 90-97DOI: (10.1016/j.joca.2007.05.009) Copyright © 2007 Osteoarthritis Research Society International Terms and Conditions

Fig. 5 Western Blot of PRG4 secreted by chondrocytes in cartilage explants containing the superficial zone under various culture conditions probed with mAb 3A4 after separation on a 3–8% gradient polyacrylamide gel. Osteoarthritis and Cartilage 2008 16, 90-97DOI: (10.1016/j.joca.2007.05.009) Copyright © 2007 Osteoarthritis Research Society International Terms and Conditions

Fig. 6 Correlation of PRG4 secretion by chondrocytes in cartilage explants containing the superficial zone over 6 days of culture to (A) the total PRG4+ cells per area in the cartilage explants, and (B) the amount of PRG4 bound at the surface of the cartilage explants, at the end of the culture duration. Shaded regions indicate levels at or below assay sensitivity. Osteoarthritis and Cartilage 2008 16, 90-97DOI: (10.1016/j.joca.2007.05.009) Copyright © 2007 Osteoarthritis Research Society International Terms and Conditions