IL-6 induces periostin production in human ACL remnants: a possible mechanism causing post-traumatic osteoarthritis

Fig. 1. Transwell co-culture system. ACL fibroblasts and chondrocytes were cultured in the upper and lower compartment, respectively. The compartments were separated by a 0.4 μm permeable membrane

Fig. 2. Immunofluorescence staining of IL-6 receptors in ACL fibroblasts. IL-6 receptors: Alexa Fluor 488-conjugated anti-rabbit secondary antibody (green). Nuclei were counterstained in blue (DAPI)

Fig. 3. The results of quantitative real-time PCR. The mean value of POSTN/GAPDH of 1mg/ml was set at 1 for clear comparisons. The expression amounts of 0, 1, 5, 50, and 500 ng/ml IL-6 were significantly different (P = 0.001 for 5 versus 50 ng/ml, P < 0.001 for the other comparisons)

Fig. 4. The results of western blot analysis regarding different IL-6 concentrations. A The representative experiment. B Quantitative analysis of the western blot analysis with Image J software (National Institutes of Health, Bethesda, Maryland, USA). IL-6 dose-dependently induced POSTN production. The mean value of POSTN/β-actin of 0 mg/ml was set at 1 for clear comparisons. ns: not significant

Fig. 5. The results of western blot analysis regarding the duration of IL-6 treatment. A The representative experiment. B Quantitative analysis of the western blot. IL-6 time-dependently induced POSTN production (P = 0.012)

Fig. 6. The results of western blot analysis regarding time from injury. A The representative experiment. B Quantitative analysis of the western blot analysis. The POSTN production was comparable between the two groups (P = 0.880)

Fig. 7. The results of western blot analysis regarding possible signaling pathways. A The representative experiment. B Quantitative analysis of the western blot analysis. Only LY294002 significantly attenuate the effect of IL-6 on POSTN production. *P < 0.05 when compared to IL-6 group

Fig. 8. Immunohistochemical POSTN staining. A The representative picture. B The average IHC score was significantly higher in the IL-6 group (P = 0.017)

Fig. 9. The periostin concentration of culture media in the co-culture system. The concentration was significantly higher in the coexistence of ACL fibroblasts and IL-6 (P = 0.018)

Fig. 10. The results of western blot analysis regarding MMP-13 and ADAMTS-4 production by chondrocytes. Coexistence of ACL and IL-6 induced more production (P = 0.048 for MMP-13 and P = 0.040 for ADAMTS-4)

Fig. 11. The possible cross-talk between ACL fibroblasts and chondrocyte. *The finding of a previous report [29]