Adipose-derived stem cells induce autophagic activation and inhibit catabolic response to pro-inflammatory cytokines in rat chondrocytes Li-Bo Jiang,

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Adipose-derived stem cells induce autophagic activation and inhibit catabolic response to pro-inflammatory cytokines in rat chondrocytes Li-Bo Jiang, Soomin Lee, Yang Wang, Qin-Tong Xu, De-Hua Meng, Jian Zhang Osteoarthritis and Cartilage Volume 24, Issue 6, Pages 1071-1081 (June 2016) DOI: 10.1016/j.joca.2015.12.021 Copyright © 2016 Terms and Conditions

Fig. 1 Characterization of ADSCs and the co-culture of ADSCs and chondrocytes. A. Surface markers of ADSCs were examined by flow cytometric analysis. The green curves indicate the result of negative control; while the red curves indicate the results of ADSCs samples with different antibodies. The overlap sections of green and red curves indicate the negative percentages of surface markers in ADSCs; while the remaining part of red curves indicate the positive percentages. B, C. The adipogenic and osteogenic differentiations of ADSCs were analyzed by Oil red O and Alizarin Red staining respectively. Typical images were shown. D. The typical images of chondrocytes and ADSCs under phase-contrast microscopy. E. Co-culture of ADSCs and chondrocytes. ADSCs were grown in the insert and chondrocytes were cultured in the bottom of transwell. Osteoarthritis and Cartilage 2016 24, 1071-1081DOI: (10.1016/j.joca.2015.12.021) Copyright © 2016 Terms and Conditions

Fig. 2 LC3-II/LC3-I and SQSTM1 expression in chondrocytes investigated by Western blotting. A, B. Chondrocytes treated or not with 10 ng/ml IL-1β in monoculture or in co-culture with ADSCs for 24 h and 48 h. The expressions of LC3-II/LC3-I and SQSTM1 were shown by Western blotting. E, F. The optical densities for the LC3-II/LC3-I and SQSTM1/β-actin were analyzed. Data were shown as mean ± CI (confidence interval), *P < 0.05, n = 6. N = 6 means 6 independent cell isolations/preparations. C, D. Chondrocytes treated or not with 1 μg/ml LPS in monoculture or in co-culture with ADSCs for 24 h and 48 h. The expressions of LC3-II/LC3-I and SQSTM1 were shown by Western blotting. G, H. The optical densities for the LC3-II/LC3-I and SQSTM1/β-actin were analyzed. Data were shown as mean ± CI, *P < 0.05, n = 6. Osteoarthritis and Cartilage 2016 24, 1071-1081DOI: (10.1016/j.joca.2015.12.021) Copyright © 2016 Terms and Conditions

Fig. 3 MDC staining and autophagic flux analysis in chondrocytes in co-culture with ADSCs. A. Chondrocytes treated or not with IL-1β/LPS in monoculture or in co-culture with ADSCs for 24 h were stained with MDC for acidic autophagic vesicles. The white arrows indicate the autophagic vesicles. B. Chondrocytes were transfected with mRFP-GFP-LC3, and then they were treated with IL-1β and co-cultured with ADSCs as described above. White arrows indicated the yellow autophagosomes which were quantified in the D, and the triangle showed the red autolysosomes which were quantified in the E. Data were shown as mean ± CI, *P < 0.05, n = 6. C. Chondrocytes were transfected with mRFP-GFP-LC3, and then they were treat with LPS and co-cultured with ADSCs as described above. White arrows indicated the yellow autophagosomes which were quantified in the F, and the red autolysosomes were quantified in the G. Data were shown as mean ± CI, *P < 0.05, n = 6. H, I. Chondrocytes treated with 0.1 μM Bafilomycin A1 and IL-1β or LPS in monoculture or in co-culture with ADSCs for 48 h. The expressions of LC3-II/LC3-I were shown by Western blotting. The optical density for the LC3-II/LC3-I was analyzed. Data were shown as mean ± CI, *P < 0.05, n = 6. Osteoarthritis and Cartilage 2016 24, 1071-1081DOI: (10.1016/j.joca.2015.12.021) Copyright © 2016 Terms and Conditions

Fig. 4 P-mTOR and mTOR expression in chondrocytes. A. Chondrocyte treated or not with IL-1β in monoculture or in co-culture with ADSCs for 48 h were analyzed by Western-blotting for p-mTOR. C. The optical density for the p-mTOR/mTOR in chondrocytes was analyzed. Data were shown as mean ± CI, *P < 0.05, n = 6. B. Chondrocyte treated or not with LPS in monoculture or in co-culture with ADSCs for 48 h. D. The optical density for the p-mTOR/mTOR in chondrocytes was analyzed. Data were shown as mean ± CI, *P < 0.05, n = 6. Osteoarthritis and Cartilage 2016 24, 1071-1081DOI: (10.1016/j.joca.2015.12.021) Copyright © 2016 Terms and Conditions

Fig. 5 The mRNA expressions of MMPs, ADAMTSs and TIMPs in chondrocytes. A. Chondrocyte treated or not with IL-1β in monoculture or in co-culture with ADSCs for 24 h were analyzed by real-time PCR for mRNA expression. Data were shown as mean ± CI, *P < 0.05, n = 6. B. Chondrocytes treated or not with LPS for 24 h in monoculture or in co-culture with ADSCs were analyzed by real-time PCR for mRNA expression. Data were shown as mean ± CI, *P < 0.05, n = 6. Osteoarthritis and Cartilage 2016 24, 1071-1081DOI: (10.1016/j.joca.2015.12.021) Copyright © 2016 Terms and Conditions

Fig. 6 The mRNA expressions of MMPs, ADAMTSs and TIMPs in chondrocytes. A. Chondrocytes treated or not with IL-1β in monoculture or in co-culture with ADSCs for 48 h were analyzed by real-time PCR for mRNA expression. Data were shown as mean ± CI, *P < 0.05, n = 6. B. Chondrocytes treated or not with LPS in monoculture or in co-culture with ADSCs for 48 h were analyzed by real-time PCR for mRNA expression. Data were shown as mean ± CI, *P < 0.05, n = 6. Osteoarthritis and Cartilage 2016 24, 1071-1081DOI: (10.1016/j.joca.2015.12.021) Copyright © 2016 Terms and Conditions