1. Restriction of spontaneous and prednisolone-induced leptin production to dedifferentiated state in human hip OA chondrocytes: role of Smad1 and β-catenin activation 
E. Charlier, O. Malaise, M. Zeddou, S. Neuville, G. Cobraiville, C. Deroyer, C. Sanchez, P. Gillet, W. Kurth, D. de Seny, B. Relic, M.G. Malaise 
Osteoarthritis and Cartilage 
Volume 24, Issue 2, Pages (February 2016)
DOI: /j.joca
Copyright © 2015 Osteoarthritis Research Society International Terms and Conditions

2. Fig. 1
Dedifferentiated and hypertrophic markers evolution during human OA hip chondrocytes dedifferentiation. Human articular chondrocytes were isolated from OA hip cartilage and cultivated in a monolayer during 14 days. (A) Images of chondrocytes morphology were acquired by phase contrast microscopy, after 1, 4 and 14 days of monolayer culture. Scale bar: 25 μm. Photos shown are representative of three independently performed experiments. (B) Total RNAs were isolated and analyzed by quantitative RT-PCR to determine the relative gene expression of Sox9, COL2A1 and COL1A1 in OA dedifferentiated (D14) compared to OA freshly isolated (D1) chondrocytes. Input amounts were normalized with the β2-microglobulin endogenous control gene (n = 5). Hypertrophic markers were analyzed (C) in whole cell lysates by western blotting experiment to test the presence of Runx-2 with specific antibody, (D) in supernatants by specific MMP-13 (n = 7) and type X collagen (n = 7) ELISAs, and (E) in total RNAs, evaluating COL10A1 relative gene expression by quantitative RT-PCR (n = 5).
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2015 Osteoarthritis Research Society International Terms and Conditions

3. Fig. 2
Leptin production during human OA chondrocyte dedifferentiation. Human OA hip chondrocytes were maintained in a monolayer for 1, 4 and 14 days in the presence or absence of 1 μM prednisolone. (A) Leptin expression was detected in chondrocytes supernatants by ELISA (n = 8). (B) Ob-R expression was analyzed by western blotting using specific antibody. (C) Leptin production by OA dedifferentiated chondrocytes in response to increasing prednisolone concentrations for 7 days (n = 3). (D) Relative gene expression of leptin was evaluated in total RNAs from dedifferentiated compared to freshly isolated chondrocytes by RT-qPCR using specific primers, and normalized to β2-microglobulin expression (n = 5).
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2015 Osteoarthritis Research Society International Terms and Conditions

4. Fig. 3
Expression of leptin in normal and OA human hip cartilage. (A) Sections from OA knee cartilage (n = 1) were analyzed by immunohistochemistry without primary antibody (Control, upper photo) or with an anti-leptin (sc-842, 1:50 dilution) antibody (lower photo). (B) Comparison between leptin staining in normal hip cartilage (n = 1) and OA hip cartilage (n = 2). Positive cells are brown; nuclei are counterstained with hematoxylin. Arrows indicate zoomed cells (insets) below. Magnification 5×. Scale bar: 250 μm.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2015 Osteoarthritis Research Society International Terms and Conditions

5. Fig. 4
TGFβ catabolic endoglin/ALK1/p-Smad1/5 pathway is activated in dedifferentiated OA chondrocytes whereas anabolic ALK5/p-Smad2 pathway is rather activated in freshly isolated chondrocytes. Human hip OA chondrocytes were cultivated in a monolayer for 1, 4 and 14 days. Expression of key protein of anabolic (A) and catabolic (B) TGFβ pathways were visualized by western blotting analysis, using specific antibodies. GAPDH and α-tubulin were used as loading control. (C) Anti-TGFβ inactivating antibody (R&D, MAB1835) was added at indicated concentrations for 24 h on dedifferentiated chondrocytes. Whole cell lysates were next analyzed by western blotting using anti-p-Smad1/5 antibody. GAPDH is used as a loading control. (D) Effect of prednisolone on p-Smad1/5 expression in dedifferentiated chondrocytes is showed by western blotting analysis.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2015 Osteoarthritis Research Society International Terms and Conditions

6. Fig. 5
Smad1 silencing downregulates leptin production in dedifferentiated OA chondrocytes. Dedifferentiated OA chondrocytes were infected with shRNA lentiviral constructs targeting Smad1. 48 h after, infected cells were treated or not with prednisolone (1 μM) during 5 days before whole cell lysates and cell supernatants collection. (A) Leptin concentration was calculated by ELISA on cellular supernatants from non infected (ctrl), empty vector- (empty), shSmad1_1- or shSmad1_2- infected dedifferentiated chondrocytes (n = 3) (B) Western blotting was performed to verify Smad1 silencing and to visualize Ob-R expression. GAPDH is used as a loading control.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2015 Osteoarthritis Research Society International Terms and Conditions

7. Fig. 6
Endoglin silencing does not affect leptin production in dedifferentiated OA chondrocytes. Dedifferentiated chondrocytes were infected with shRNA lentiviral constructs targeting endoglin. 48 h after, infected cells were treated or not with prednisolone (1 μM) during 5 days before whole cell lysates and cell supernatants collection. (A) Leptin concentration was calculated by ELISA on cellular supernatants from non infected (ctrl), empty vector- (empty), shENG_1- or shENG_2- infected dedifferentiated chondrocytes (n = 3) (B) Western blotting was performed to verify endoglin silencing and to visualize Ob-R expression. GAPDH is used as a loading control.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2015 Osteoarthritis Research Society International Terms and Conditions

8. Fig. 7
Wnt/β-catenin pathway is activated in dedifferentiated OA chondrocytes. Human articular chondrocytes were isolated from OA hip cartilage and cultivated with or without prednisolone, in a monolayer during 14 days. Whole cell lysates were performed at indicated time points. (A) β-catenin, (B) β-catenin active form and GSK3 inactive form (p-GSK-3α/β (Ser21/9)) were determined by western blotting using specific antibodies. GAPDH was used as a loading control.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2015 Osteoarthritis Research Society International Terms and Conditions

9. Fig. 8
β-catenin silencing downregulates leptin production in dedifferentiated OA chondrocytes. Dedifferentiated OA chondrocytes were infected with shRNA lentiviral constructs targeting β-catenin. 48 h after, infected cells were treated or not with prednisolone (1 μM) during 5 days before whole cell lysates and cell supernatants collection. (A) Leptin concentration was calculated by ELISA on cellular supernatants from non infected (ctrl), empty vector- (empty), shβ-catenin_1- or shβ-catenin_2- infected dedifferentiated OA chondrocytes (n = 3) (B) Western blotting was performed to verify β-catenin silencing and to visualize Ob-R expression. GAPDH is used as a loading control.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2015 Osteoarthritis Research Society International Terms and Conditions