1. AG-041R, a novel indoline-2-one derivative, stimulates chondrogenesis in a bipotent chondroprogenitor cell line CL-1 
Hidetomo Kitamura, D.V.M., Ph.D., Makoto Okazaki, Ph.D. 
Osteoarthritis and Cartilage 
Volume 13, Issue 4, Pages (April 2005)
DOI: /j.joca
Copyright © 2005 OsteoArthritis Research Society International Terms and Conditions

2. Fig. 1 Chemical structures of CCK2/gastrin receptor antagonists.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
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3. Fig. 2
Chondrogenic effect of AG-041R on chondroprogenitor cell lines. (A, B) Histological change in CL-1 induced by AG-041R. A, control (0.1% DMSO); B, AG-041R 10μmol/L. AG-041R induced dominant chondrogenesis and marked suppression of adipogenesis in CL-1. Treatment for 7 days after confluence. Double staining of alcian blue pH 1.0 and oil red O. Counter-staining: 0.3% nuclear fast red. (C) Alcian blue intensity was measured after 7 days culture in the presence of AG-041R or vehicle control (0.1% DMSO). AG-041R induced drastic increase of alcian blue intensity in CL-1. The chondrogenic effect of AG-041R revealed in a dose-dependent manner. *: P<0.05, **: P<0.01: Significant difference from control (Student's t test).
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
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4. Fig. 3
The effect of AG-041R on the growth of CL-1. AG-041R significantly suppressed growth of CL-1 cells at the concentration of 0.1–10μmol/L in a dose-dependent manner similar to TGF-β1. Control: α-MEM containing 0.1% DMSO and 20% FCS. Medium: α-MEM containing 20% FCS. Mean+SD values. *: P<0.05, **: P<0.01 (significant difference from control, Student's t test). ##: P<0.01 (significant difference from medium, Student's t test).
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
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5. Fig. 4
Effect of AG-041R on synthesis of cartilage matrix proteins in CL-1. Expression of mRNA of cartilage matrix proteins, including collagen type II and aggrecan, was examined by Northern blotting in control (0.1% DMSO) or AG-041R (10μmol/L) treated CL-1 cells at 1, 3 and 7 days after addition. Messenger RNA of these proteins was obviously increased by the AG-041R treatment.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
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6. Fig. 5
Chondrogenic effects of other CCK2/gastrin receptor antagonists on CL-1. Chondrogenic effects of gastrin/CCK2 receptor antagonists L-365,260R, YM-022 or AG-041S in CL-1. At confluence, L-365,260R, YM-022 (0.1, 1.0, 10μmol/L), AG-041R, AG-041S (1, 10μmol/L) or 0.1% DMSO (vehicle control) was added into the culture media of CL-1 cells in 24-well plates. Alcian blue intensity was measured after 7 days culture in the presence of L-365,260R, YM-022, AG-041R, AG-041S or 0.1% DMSO. Neither L-365,260R nor YM-022 significantly increased the alcian blue intensity of CL-1 (upper panels). AG-041S significantly suppressed the alcian blue intensity of CL-1 at the concentrations of 1 and 10μmol/L (lower panel). Mean+SD values. **: P<0.01 (significant difference from control, Student's t test).
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
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7. Fig. 6
Induction of TGF-β proteins in CL-1 by AG-041R. TGF-β concentrations (A, B: TGF-β1; C, D: TGF-β2) in culture media of control (0.1% DMSO) or AG-041R (10μmol/L) treated CL-1. (A, C) Concentration of active TGF-β. (B, D) Concentration of total TGF-β. For this assay, samples were activated by 0.01mol/L HCl. TGF-β contents (E: TGF-β1; F: TGF-β2) in the cell layer of the control (0.1% DMSO) or AG-041R (10μmol/L) treated CL-1. TGF-β contents were normalized with DNA content in cell layer samples. Mean+SD values. *: P<0.05, **: P<0.01 (significant difference from control, Student's t test).
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
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8. Fig. 7
Blocking of TGF-β signaling by neutralizing antibody in AG-041R treated CL μg/mL neutralizing antibody MAS-603 almost completely blocked the stimulation of proteoglycan synthesis by AG-041R. MOPC-31C, a control murine IgG1 did not block the chondrogenic effect of AG-041R in CL-1. Mean+SD values. ##: P<0.01 (significant difference from control, Student's t test). **: P<0.01 (significant difference from AG-041R, Student's t test).
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
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9. Fig. 8
Effects of inhibitors of TGF-β activation on chondrogenic effect of AG-041R in CL-1. (A) Inhibition of TGF-β activation in CL-1 by aprotinin, a serine protease inhibitor or M6P, an IGF-II/M6P receptor antagonist. Aprotinin drastically suppressed the chondrogenic activity of AG-041R in CL-1 (2TIU/mL<). M6P did not inhibit the effect of AG-041R. (B) Inhibition of furin, a prohormone convertase, by dec-RVKR-cmk. This inhibitor suppressed the chondrogenic effect of AG-041R (5μmol/L<). Mean+SD values. **: P<0.01 (significant difference from AG-041R, Student's t test) ##: P<0.01 (significant difference from control, Student's t test).
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
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10. Fig. 9
Activation of Erk (p44/42) and p38 MAP kinases by AG-041R in CL-1. (A) Erk (p44/42) MAP kinase activation. Erk (p44/42) activation was examined by Western blotting in CL-1 cells treated with control (0.1% DMSO), AG-041R (10μmol/L) or TGF-β1(1ng/mL, 0.1% DMSO) at 1, 3, 5, 10, 30, 60 and 120min after the addition. Ten percent of FCS in medium was used as a positive control at 5min after the addition. AG-041R intensely activated Erk (p44/42) MAP kinase from 1 to 30min. TGF-β1 activated Erk (p44/42) MAP kinase from 1 to 5min. All the culture medium contains 0.1% DMSO. (B) p38 MAP kinase activation. p38 MAP kinase activation was examined at 30min after AG-041R addition. As positive control, 0.7mol/L NaCl was used. AG-041R mildly activated p38 MAP kinase. TGF-β1 did not activate p38 MAP kinase. All the culture medium contains 0.1% DMSO.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
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11. Fig. 10
Effect of MAP kinase inhibitors on the chondrogenic effect of AG-041R in CL-1. In control medium (0.1% DMSO in 10% FCS/α-MEM), PD98059 simulated adipogenesis in CL-1. SB did not alter either chondrogenesis or adipogenesis. AG-041R (2μmol/L) increased alcian blue positive cells and suppressed oil red O positive cells. PD98059 increased alcian blue stainability mildly and oil red O positive cells in the presence of AG-041R. SB canceled stimulated chondrogenesis by AG-041R with no effect on adipogenesis suppression induced by AG-041R. TGF-β1 (10ng/ml) also, increased alcian blue positive cells and suppressed oil red O positive cells. PD98059 enhanced the chondrogenic effect of TGF-β1. On the other hand, SB canceled the chondrogenic effect of TGF-β1. Neither PD98059 nor SB98059 changed adipogenesis suppression induced by TGF-β1. Alcian blue pH 1.0 and oil red O. One week culture. All the culture medium contains 0.2% DMSO.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
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