Fig. 5: Blockage of CNX protects CAIA mice from cartilage degradation and development of severe arthritis.

a Representative IF staining images show abundant presence of collagen fibres containing collagen type III (Col III), collagen type I (Col I) and fibronectin in cartilage ECM. Collagen disulfide bonds are chemically reduced using TCEP which can be detected by staining with OX133 antibodies or left untreated (UT). Scale bar, 5 μm. b Representative images and (c) quantification graph show decreased matrix degradation activity in primary fibroblasts isolated from OA patients after 48 h incubation with 5ug anti-CNX antibodies (ab22595, abcam) as compared to those treated with isotype control antibodies (ab37415, abcam). Data correspond to the mean ± SEM and representative data for three independent experiments. Each data point represents the total degraded area (μm) per nuclear per well. ***, p < 0.001 (p = 0.0003) (Mann–Whitney test, two-tailed p-value). d Antibody treatment scheme. Mice were intraperitoneally injected with 2 mg anti-collagen II antibodies (Chondrex Inc.) on day 0, followed by 50 μg lipopolysaccharide (LPS) injection on day 3. Antibody treatment was subsequently administered at 25 μg of antibody per mice on the same day and the treatment was repeated every two days. A total of four antibody treatments was given for each treated mouse. e Representative photographs of CAIA mice treated with anti-CNX antibodies or isotype antibodies at day 10. f Paw thickness measurement in CAIA animals after injecting with anti-CNX antibodies. Data represent mean ± SEM, n = 5 mice injected with isotype antibody and n = 4 mice injected with anti-CNX antibody. ****, p < 0.0001 (day 7 and day 10) (Two-way ANOVA test). g Clinical scores of CAIA mice at day 10 following treatment with isotype control antibodies (n = 5) or anti-CNX (n = 4) antibodies. Data are mean ± SEM. *, p < 0.05 (p = 0.0238) (Mann–Whitney test, two-tailed p-value). h, i Representative histological images (h) and quantification (i) of Alcian blue (AB) staining (arrow bars) in CAIA mice treated with isotype control (n = 5) or anti-CNX antibodies (n = 4). Individual data points represent average of positive staining area per mm² articular cartilage from two individual metacarpophalangeal joints of each animal. Data are shown as mean ± SEM. *, p < 0.05 (p = 0.0266) (Mann–Whitney test, two-tailed p-value). j Representative IF images show co-staining of anti-CNX antibodies (arrow heads) and VVL in CAIA mice injected with anti-CNX antibodies (n = 4) or isotype control antibodies (n = 5) at day 10. B bone, S synovium. Scale bar, 50 μm.