SOX9 Antibody - #AF6330

FIGURE 5 Pathogenic FLS EVPs impair chondrogenic differentiation of mesenchymal stem cells. (A) Schematic diagram of EVP stimulation on mouse BMSCs isolated from the femoral bone marrow cavity of mice. (B) Internalization of EVPs by BMSCs. Scale bar = 50 µm. (C) Expression of chondrogenic differentiation-related genes in chondrogenesis-induced BMSCs after 7 days of treatment with different EVPs. (D) Representative Western blot images of COL10A1, the marker of chondrocyte hypertrophy. (E, F) Representative images of alcian blue staining and SA-β-gal staining in BMSCs after 7 days of stimulation with different EVPs. Scale bar = 1 mm and 200 µm separately. (G) Schematic diagram of section staining observation after inducing BMSCs to form chondrocyte pellets for 21 days while simultaneously stimulating with different EVPs. (H, I) Representative images of SOX9 fluorescence staining, safranin O (SO) staining, alcian blue (AB) staining and toluidine blue (TB) staining of BMSC-differentiated chondrocyte pellet sections. Scale bar = 50 µm. (J) Schematic diagram of EVP treatment on mouse ADSCs isolated from the iWAT of mice. (K) Internalization of EVPs by ADSCs. Scale bar = 50 µm. (L, M) Representative images of SA-β-gal staining and alcian blue staining of ADSCs after 7 days of chondrogenic induction and treatment with different EVPs. (N) Representative images of SOX9 staining in sections of chondrocyte pellets formed by ADSC after 21-day induction.

Fig. 4 HS suppresses inflammation and promotes chondrogenesis in CCO while enhancing integrin β1 expression. (A) Immunofluorescence staining for iNOS, Arg-1, CD86, and CD206 was performed after one day of co-culture of HS hydrogel with BMDM macrophages for M1 macrophage induction, utilizing DAPI (blue) for nuclei, specific staining (red) for iNOS, CD86 and specific staining (green) for CD206, Arg-1. (B) Quantitative analysis of Figure A. All three types of hydrogels can promote the polarization of macrophages toward the M2 phenotype. (C) SO, and AB staining after four days of co-culture of HS hydrogel with BMSCs for cartilage induction. HS600 can accumulate a greater amount of cartilage matrix. (D) Quantitative analysis of mRNA transcripts for COL2 and SOX9. The expression of cartilage genes is higher in the HS600 group. (E) Fluorescence staining for COL2 and SOX9, utilizing rhodamine phalloidin (red) for F-actin, DAPI (blue) for nuclei, and specific staining (green) for COL2 and SOX9. (F) Quantitative analysis of Figure E. The expression of cartilage-related proteins is higher in the HS600 group. (G) Fluorescence staining for β1, using rhodamine phalloidin (red) for F-actin, DAPI (blue) for nuclei, and specific staining (green) for β1. (H) Quantitative analysis of Figure G. The expression of integrin β1 protein is higher in the HS600 group. (I) Staining of F-actin (green) and cell nuclei (blue) after BMSCs were seeded on the hydrogel surface for 1 day. The morphology of BMSCs in the HS group can exhibit normal spreading. (J) Immunofluorescence staining of COL2, and β1 in CCO co-cultured with HS600. (K) Quantitative analysis of Figure J. After co-culturing HS600 with CCO, the expression of cartilage markers and the adhesion factor β1 are enhanced. All experiments were repeated three times. ns p > 0.05, *0.01 

Fig. 7 The chondrogenic differentiation of iPSCs with different Li+ ions concentration in 14 days. a Immunofluorescence of chondrocytes specified proteins (COL II, Aggrecan, and SOX9) and hypertrophic specified protein (COL X and MMP13). b The average fluorescence intensity of COL II, Aggrecan, SOX9, MMP13, and COL X proteins in each group, n = 5. c The relative genes expression of chondrogenic differentiation cultured with Li+ ions, n = 3. d The relative genes expression of hypertrophic differentiation cultured with Li+ ions, n = 3. Data presented as mean ± SEM. Scale bar = 200 μm. CTR: MCDM without any Li+ ions. *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 8. GPX4 inhibitor RSL3 substantially attenuates VK2 protection of chondrocytes. (A-I) WB results for GPX4, NFκB, pMAPK/MAPK, Aggrecan, CollagenⅡ, SOX9, MMP3, MMP13 and Tubulin; (J) Quantitive analysis of relative Glutathione Peroxidase Activity; (K-L) GSH contents and ratio of GSH/GSSG; (M) Quantitative results of MDA content assay.