1. The orphan G-protein coupled receptor RDC1: evidence for a role in chondrocyte hypertrophy and articular cartilage matrix turnover 
S.W. Jones, Ph.D., S.M.V. Brockbank, B.Sc., M.L. Mobbs, B.Sc., N.J. Le Good, B.Sc., S. Soma-Haddrick, Ph.D., A.J. Heuze, B.Sc., C.J. Langham, B.Sc., D. Timms, Ph.D., P. Newham, Ph.D., M.R.C. Needham, B.Sc. 
Osteoarthritis and Cartilage 
Volume 14, Issue 6, Pages (June 2006)
DOI: /j.joca
Copyright © 2006 OsteoArthritis Research Society International Terms and Conditions

2. Fig. 1
RDC1 expression. Immunohistochemical analysis of RDC1 expression in recombinant RDC1. HEK cell line, compared to an untransfected HEK293 cell line. Cells were stained with primary anti-RDC1 Lifespan antibody (1:50) or rabbit IgG control (Lifespan) and secondary anti-rabbit Alexa488 (Molecular Probes) and visualised by confocal microscopy.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
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3. Fig. 2
Ca2+ response to RDC1 agonists in RDC1 HEK293 cell lines. RDC1 HEK293, RDC1 Gα16 HEK293 and control HEK293 cells were stimulated with compound agonists at 1, 3, 10, 30, 100 and 300μM. Values are mean maximal responses±(n=3). The transient Ca2+ response was detected by FLIPR. (a) Activity of RDC1 agonist compound 12 in RDC1. HEK293 and untransfected HEK293 cells. (b) Comparison of potency of compound agonists 1, 5, 8 and 12.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
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4. Fig. 3
RDC1 compound agonists exhibit partial Gi coupling. (a) Effect of compound agonists 1, 5, 8 and 12 (10–100μM) on cAMP stimulation in HEK293 RDC1 cells. Bars represent mean±s.e.m. (b) Effect of compound agonists on the inhibition of the forskolin cAMP induction. Intracellular cAMP was measured using a BioTrak kit (Amersham). Bars represent mean±s.e.m. *=Significantly different (P<0.05) from the forskolin cAMP response. (c) Inhibition of compounds 5 and 12 Ca2+ response in HEK293 RDC1 cells following deactivation of Gq proteins by overnight incubation with pertussis toxin (100ng/ml). Bars represent mean±s.e.m. (n=6). *=Significantly different from untreated control HEK293 RDC1 cells.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2006 OsteoArthritis Research Society International Terms and Conditions

5. Fig. 4
RDC1 is expressed and is active in primary chondrocytes. (a) Human primary chondrocytes were prepared from a normal knee and the Ca2+ response to compounds 1, 5, 8 and 12 (30–300μM) measured by FLIPR. Values are mean maximal response±(n=3). (b) Primary chondrocytes stained using the polyclonal RDC1 antibody or IgG control (1:200).
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2006 OsteoArthritis Research Society International Terms and Conditions

6. Fig. 5
RDC1 is highly expressed in cartilage tissue, but is not differentially expressed in OA. (a) Biodistribution of RDC1 mRNA expression. (b) Expression of RDC1 in human normal (n=9) and osteoarthritic (n=7) cartilage samples. Bars represent mean±s.e.m. RDC1 expression was determined by real-time PCR using Taqman primers and probes and normalised to the housekeeping gene 18S.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2006 OsteoArthritis Research Society International Terms and Conditions

7. Fig. 6
Effect of RDC1 agonist on cartilage explant gene expression. Cartilage explant discs were prepared from three normal (solid bars) and three OA (hatched bars) human knees and incubated for 24h at 37°C with either media control, RDC1 agonist compound 5 (100μM) or inert compound (100μM). Total RNA was extracted and gene expression of (a) MMPs, (b) VEGF, and (c) BMP2 was determined. (d) Differential effect of RDC1 agonist compound 5 on Col2 and aggrecan expression in OA and PM explant. Gene expression was determined by quantitative real-time PCR from 25ng total RNA using Taqman probes (Applied Biosystems). Bars represent mean fold change from control value±s.e.m. (n=3). *=Significantly different from basal.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
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8. Fig. 7
Effect of RDC1 agonist on normal cartilage explant total MMP activity. Normal cartilage explant discs were prepared from three PM donors and treated for 24h with RDC1 agonist compound 5 (100μM) or media control. Supernatant was then removed and assayed for total MMP activity using ELISA kit (info). Bars represent mean±s.e.m. (n=20).
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2006 OsteoArthritis Research Society International Terms and Conditions

9. Fig. 8
Effect of RDC1 agonist on MMP1 and Col2 expression in HEK293 cell lines. RDC1 HEK293 and control HEK293 cells were stimulated with RDC1 agonist compound 5 (100μM) or with media control for 24h. MMP1 and Col2 mRNA expression was determined by real-time PCR using Taqman probes. Bars represent mean±s.e.m. (n=3). *=Significantly different from media control (P<0.01).
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2006 OsteoArthritis Research Society International Terms and Conditions

10. Fig. 9
Validation that the compound agonists are acting through RDC1. Impact of RDC1 siRNA. (a) Percentage knockdown of RDC1 following transfection of RDC1 siRNA at 100nM into HEK293 cells and at 1nM in primary chondrocytes using Lipofectamine 2000 (Invitrogen) and Atugen lipid, respectively. (b) Impact of RDC1 siRNA (100nM) transfection on FLIPR response to agonist compound in HEK293 RDC1 cell line. (c) Impact of RDC1 siRNA (1nM) transfection on RDC1-regulated genes in primary chondrocytes. Bars represent mean±s.e.m. (n=3). *=Significantly different from non-targeting siRNA control (P<0.01).
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2006 OsteoArthritis Research Society International Terms and Conditions