1. Resistin promotes CCL4 expression through toll-like receptor-4 and activation of the p38-MAPK and NF-κB signaling pathways: implications for intervertebral disc degeneration 
Z. Li, X. Wang, H. Pan, H. Yang, X. Li, K. Zhang, H. Wang, Z. Zheng, H. Liu, J. Wang 
Osteoarthritis and Cartilage 
Volume 25, Issue 2, Pages (February 2017)
DOI: /j.joca
Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

2. Fig. 1
The expression of resistin and CCL4 in human NP tissue and the resistin-induced macrophage migration. RT-PCR showed that the mRNA levels of resistin and CCL4 in the NP tissue were significantly higher compared with the AF tissue (A, B). Representative MRI images of different degenerated discs are shown in C. The mRNA expression levels of resistin and CCL4 in severely degenerated NP tissue [Grade VI (n = 4), V (n = 5)] were significantly higher compared to the mildly degenerated NP tissue [Grade I (n = 2), II (n = 3), III (n = 5)] (D, E). The conditioned medium of rat NP cells treated with resistin promoted chemotactic migration of macrophages, which was completely blocked when the macrophages were pretreated with J (F). Resistin-dependent macrophage migration was partially blocked by CCL4-specific siRNA (G). (*p < 0.05 compared with the control group).
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

3. Fig. 2
Resistin-induced CCL4 expression in the NP cells. RT-PCR demonstrated that resistin significantly increased the mRNA levels of CCL4, which peaked at 8 h compared to the control (A). CCL4 mRNA expression was dose-dependent (B). An ELISA showed that induction of CCL4 protein secretion by resistin was time-dependent in the NP cells (C). Immunofluorescence microscopy showed that CCL4 expression was elevated after resistin treatment. (*p < 0.05 compared with the control group).
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

4. Fig. 3
TLR-4 is involved in the induction of CCL4 by resistin. A luciferase reporter assay showed that resistin significantly increased CCL4 promoter activity, which peaked at 8 h compared to the control (B). Resistin significantly enhanced CCL4 promoter activity in a dose-dependent manner (C). CCL4 promoter activity was significantly decreased by TLR-4 inhibition (C34, Tocris) compared to the control (D). RT-PCR showed TLR-4 siRNA significantly decreased the mRNA level of TLR-4 (E). RT-PCR showed that C34 and TLR-4 siRNA significantly decreased the mRNA levels of CCL4 (F). An ELISA showed that CCL4 protein in the conditioned medium was also decreased by C34 and TLR-4 siRNA compared to the controls (G). The co-immunoprecipitation data showed that, compared to the IgG and untreated group (lane IgG and C), the binding of resistin to TLR4 was significantly increased (lane R) after treatment with resistin (H). (*p < 0.05 compared with the control group).
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

5. Fig. 4
Resistin activates the p38-MAPK and NF-κB signaling pathways. Western blot analysis showed there was a rapid increase in the phosphorylation levels of p38 and NF-κB after resistin treatment. The activation of p38 peaked at 5–15 min before declining and staying elevated until 24 h (B), while activation of NF-κB peaked at 24 h (A). Immunofluorescence microscopy showed that p65 and phospho-p65 expression was elevated after resistin treatment, which was blocked by the NF-κB inhibitor, SM7368.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

6. Fig. 5
Inhibition of the p38-MAPK and NF-κB signaling pathways blocks the induction of CCL4 by resistin. A luciferase reporter assay showed that p38 inhibition (SB203580) and NF-κB inhibition (SM7368) significantly abolished the resistin-induced up-regulation of the activity of the CCL4 promoter (A). RT-PCR and Western blotting also showed that p38 inhibition and NF-κB inhibition significantly abolished the resistin-induced up-regulation of the gene (B) and protein expression of CCL4 (C). Immunofluorescence microscopy demonstrated that the inhibition of p38-MAPK and NF-κB signaling antagonized the resistin induced up-regulation of CCL4 (D). (*p < 0.05 compared with the control group).
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

7. Fig. 6
Lentivirus-mediated knockdown of p65 blocks the induction of CCL4 by resistin. The expression of p65 was significantly knocked down using short hairpin p65 (shp65) lentivirus transduction (A, B). The shp65 lentivirus significantly blocked the resistin-induced up-regulation of CCL4 mRNA levels (C). A promoter deletion assay was performed and a series of deletion fragments of the CCL4 promoter, from −1157/+400 bp to −325/+400 bp, were linked to the pGL-3 luciferase reporter (D). CCL4 promoter constructs of different lengths were detected. The CCL4 promoter activity was dramatically attenuated after deletion of the −796/−560 bp region (E). A ChIP assay was performed to investigate whether resistin regulation was through an enhanced interaction between p65 and the CCL4 promoter. The PCR products of input and the DNA precipitated with the anti-p65 antibody indicating that interaction between p65 and the CCL4 promoter was enhanced by resistin (F). Normal IgG was used as a negative control. (*p < 0.05 compared with the control group). A proposed model of the relationship between resistin and CCL4 in the NP cells (G).
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions