Figures and data
The severe cartilage damage in human HA.
(A) Human cartilage samples in the tibial plateau were obtained from patients with OA and HA. (B) MRI imaging of the knee joint in patients with HA and OA. Red arrow indicates the wear area. (C) Representative images of ABH/OG staining for HA and OA cartilage. (D) The degree of cartilage degeneration was quantified according to the OARSI score. (E) Representative IHC staining of COL2A1 and MMP13. (F) Quantification of the proportion of COL2A1 and MMP13 positive regions in human cartilage. (G) Representative IHC staining of DNMT1 and DMMT3A. (H) Quantification of the proportion of NMT1 and DMMT3A positive cells in human cartilage. Red arrows indicate positive cells. Scale bar: 100 μm. Data were presented as means ± SD and analyzed by 2-tailed unpaired parametric Student’s t test, **P < 0.01, ***P < 0.001.
Genome-wide DNA methylation profile in HA cartilage and biological functions of DMR-Related genes.
(A) Distribution of methylation level density of CpGs. Note: X = degree of methylation; Y = the CpG site density corresponding to the level of methylation. (B) Principle component analysis of DNA methylation data. (C) Heatmap shows the 700 signifricant DMRs between HA and OA. (D) Enriched GO terms for DMR-related genes. (E) KEGG enrichment analysis of DMR-related genes. (F) Representative IHC staining of TNXB. Red arrows indicate positive areas. Scale bar: 100 μm. (G) Quantification of the proportion of TNXB positive regions in human cartilage. Data were presented as means ± SD and analyzed by 2-tailed unpaired parametric Student’s t test, ***P < 0.001.
TNXB expression is drastically reduced in HA mouse cartilages.
(A) Representative images and (B) Quantitative analysis of Toluidine blue staining for knee sections of F8−/− mice at 4 and 8 weeks following injury. Representative IHC staining of (C) Col2a1, (E) Mmp13, (G) Tnxb, and (I) Dnmt1 in F8−/− mice at 4 and 8 weeks post injury. Quantification of the proportion of (D) Col2a1, (F) Mmp13, (H) Tnxb and (J) Dnmt1 positive regions. Scale bar: 100 μm. Data were presented as means ± SD and analyzed by 2-tailed unpaired parametric Student’s t test, ***P < 0.001, n ≥ 3 in each group.
knockdown of Tnxb in chondrocytes induces extracellular matrix degradation and accelerates the progression of HA.
(A) qPCR and (B) Western blotting analysis for Tnxb in mouse chondrocytes treated with siRNA-NC or siRNA-Tnxb. (C) Corresponding quantification analysis of Tnxb protein. Quantification of mRNA levels for (D) Mmp13 andCol2a1 in Tnxb-KD chondrocytes. (E) Western blot and (F) corresponding quantification analysis forMmp13 and Col2a1 protein. (G) Representative immunofluorescence images of Col2a1 and Mmp13 expression in Tnxb-KD chondrocytes. (H) Quantification of Col2a1 and Mmp13 fluorescence intensity. Representative images of (I) Toluidine blue staining and (K) IHC staining of Mmp13 and Col2a1 for knee sections of F8−/− mice at 4 weeks after Intra-articular injection of AAV-shTnxb. (J) Quantitative detection of the area of the tibial cartilage area. (L) Quantification of the proportion of Col2a1 and Mmp13 positive regions.Red arrow indicates the wear area. Scale bar: 100 μm. Data were presented as means ± SD and analyzed by 2-tailed unpaired parametric Student’s t test, *P < 0.05, **P < 0.01, ***P < 0.001, n ≥ 3 in each group.
Tnxb knockdown increases apoptosis in chondrocytes and HA cartilages.
(A) Representative images of TUNEL staining in Tnxb-KD chondrocytes (B) Quantification for TUNEL positive cells. (C-F) Western blot and corresponding quantification analysis for Bax, Cleaved-Caspase3, and Bcl-2. (G) TUNEL staining for apoptosis in articular cartilage from Tnxb-KD HA mice. (H) Quantification for TUNEL positive cells in articular cartilage. Representative IHC staining of Bax (I), Bcl-2 (K), and p-Akt1 (M) in articular cartilage from Tnxb-KD HA mice. Corresponding quantification of the proportion of (J) Bax, (L) Bcl-2 and (N) p-Akt1 positive regions. Red arrows indicate positive cells. Scale bar: 100 μm. Data were presented as means ± SD and analyzed by 2-tailed unpaired parametric Student’s t test, **P < 0.01, ***P < 0.001, n ≥ 3 in each group.
Treatment with AKT agonist decreases apoptosis in TNXB-KD chondrocytes.
(A) Western blot analysis for Col2a1, Mmp13, Bax, Bcl-2, p-Akt1 and Akt1 in Tnxb-KD chondrocytes treated with SC79 (0 or 10μM) for 24 hours. (B-F) Corresponding quantification of these proteins. (G) Schematic of the role of TNXB in the pathogenesis of HA cartilage degeneration. Data were presented as means ± SD and analyzed by 2-tailed unpaired parametric Student’s t test or one-way ANOVA with Tukey’s multiple comparisons test, *P < 0.05, **P < 0.01, ***P < 0.001, n ≥ 3 in each group.
