L. J. Sandell, Ph. D. , X. Xing, M. D. , C. Franz, M. A. , S

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Exuberant expression of chemokine genes by adult human articular chondrocytes in response to IL-1β L.J. Sandell, Ph.D., X. Xing, M.D., C. Franz, M.A., S. Davies, Ph.D., L.-W. Chang, Ph.D., D. Patra, Ph.D. Osteoarthritis and Cartilage Volume 16, Issue 12, Pages 1560-1571 (December 2008) DOI: 10.1016/j.joca.2008.04.027 Copyright © 2008 Osteoarthritis Research Society International Terms and Conditions

Fig. 1 RT-PCR validation of microarray. Chondrocytes from normal human knee cartilage were treated with 10ng/ml of IL-1β for 24h. RNA extracted from these cells was used for microarray analysis as well for RT-PCR validation. Control cells (C) received vehicle only. Total RNA samples from treated and untreated cells were analyzed by RT-PCR with the primers indicated in Table I. GAPDH was used as reference for gel loading. Quantification of individual bands was done by NIH IMAGE J software analysis with PCR in the linear range of product (Table II). Osteoarthritis and Cartilage 2008 16, 1560-1571DOI: (10.1016/j.joca.2008.04.027) Copyright © 2008 Osteoarthritis Research Society International Terms and Conditions

Fig. 2 Dose response of chemokines and matrix molecules to IL-1β. (A) Chondrocytes from normal human knee cartilage were treated with 0.01, 0.1 and 1ng/ml of IL-1β for 24h. Control cells (C) received vehicle only. RT-PCR was done with indicated primers and GAPDH was used as reference. (B) Quantitation of RT-PCR analysis with gene expression normalized to GAPDH expression using NIH IMAGE J software. Pixel intensity is given in arbitrary units (AU). Osteoarthritis and Cartilage 2008 16, 1560-1571DOI: (10.1016/j.joca.2008.04.027) Copyright © 2008 Osteoarthritis Research Society International Terms and Conditions

Fig. 3 Time course of response of chemokines and matrix molecules to IL-1β. Chondrocytes from preserved areas of cartilage from knees of OA patients were treated with 0.1ng/ml of IL-1β for 0, 1, 4, 8, 12, and 24h. Control cells (C) received vehicle only. RT-PCR was done with indicated primers with GAPDH used as reference. Osteoarthritis and Cartilage 2008 16, 1560-1571DOI: (10.1016/j.joca.2008.04.027) Copyright © 2008 Osteoarthritis Research Society International Terms and Conditions

Fig. 4 Response of normal and OA chondrocytes to IL-1β. Chondrocytes from normal human knee cartilage and preserved areas of OA knee cartilage were exposed to 0.1ng/ml of IL-1β for 24h. Control cells (C) received vehicle alone. Genes which showed an increase in expression from OA chondrocytes without addition of IL-1β are denoted by *. Osteoarthritis and Cartilage 2008 16, 1560-1571DOI: (10.1016/j.joca.2008.04.027) Copyright © 2008 Osteoarthritis Research Society International Terms and Conditions

Fig. 5 Reversal of IL-1β-induced phenotype by TGF-β1. Chondrocytes from normal knee cartilage and preserved areas of OA knee cartilage were exposed to 0.1ng/ml of IL-1β for 24h. Control cells (C) received vehicle alone. After 24h, medium containing IL-1β was removed and fresh medium containing 10ng/ml of TGF-β1 was added for 48h. RNA samples were analyzed by RT-PCR with indicated primers. GAPDH was used as reference for gel loading. Lanes denoted as (IL-1β) indicate that cells were treated with IL-1β for 24h and were not treated with TGF-β1; lanes denoted as TGF-β1 (IL-1β) indicate cells received TGF-β1 after removal of IL-1β; ^ indicates that the bands were too low to quantify; ∼ indicates no change. Osteoarthritis and Cartilage 2008 16, 1560-1571DOI: (10.1016/j.joca.2008.04.027) Copyright © 2008 Osteoarthritis Research Society International Terms and Conditions

Fig. 6 Effect of FGF-18 on IL-1β-induced phenotype. Chondrocytes from preserved areas of OA knee cartilage were treated with 0.01ng/ml of IL-1β for 24h. At 24h, 200ng/ml of FGF-18 was added and cultured for another 24h. Control cells (C) received vehicle alone. RNA samples were analyzed by RT-PCR with indicated primers. GAPDH was used as reference for gel loading. Osteoarthritis and Cartilage 2008 16, 1560-1571DOI: (10.1016/j.joca.2008.04.027) Copyright © 2008 Osteoarthritis Research Society International Terms and Conditions

Fig. 7 Effect of IL-1β on cartilage explants in culture. Cartilage explants from the preserved areas of knees of OA patients were treated with 10ng/ml of IL-1β for 24h. Control tissue (C) received vehicle alone. RNA samples were analyzed by RT-PCR with indicated primers. GAPDH was used as reference for gel loading. Osteoarthritis and Cartilage 2008 16, 1560-1571DOI: (10.1016/j.joca.2008.04.027) Copyright © 2008 Osteoarthritis Research Society International Terms and Conditions