Anti collagen 2

Immunofluorescence and immunohistochemistry assays were performed as previously described [2 (link), 10 (link)]. The following primary antibodies were used: anti-Cx43 (Sigma-Aldrich, C6129), anti-collagen II (Invitrogen, Thermo Fisher Scientific, MA5-12789), anti-Ki-67 (BD, 550609), anti-Twist-1 (sc-81417) and anti-NF-κB (sc-8008) from Santa Cruz Biotechnology. Goat anti-rabbit FITC-conjugated (F-2765, Invitrogen, Thermo Fisher Scientific) and goat anti-mouse Alexa 594-conjugated (A-11032, Invitrogen, Thermo Fisher Scientific) secondary antibodies were used.

SW1353 cells were seeded at a density of 5 × 10³ per well in 24-well plates. Cells were washed by PBS for three times and fixed with 4% paraformaldehyde for 20 minutes at room temperature. The cells were then incubated with blocking solution (0.2% powdered milk, 1% BSA, and 0.01% Triton X-100). After incubation for 30 min at room temperature, the cells were treated with anti-collagen II (Invitrogen, 1:5,00), Aggrecan (1:500, Santa Cruz Biotechnology, Inc.) overnight at 4°C. Cells were washed with PBS and staining with secondary antibody (1:400, Santa Cruz Biotechnology, Inc.) for 1 h at 37°C. To visualize the nuclei, 4ʹ, 6-diamidino-2-phenylindole (DAPI, Sigma) was added for 10 min, and the cells were observed under a fluorescence microscope (Zeiss).

The knee joints were fixed in 4% paraformaldehyde and decalcified with 20% ethylene diamine tetraacetic acid (EDTA). The tissues were embedded in paraffin and cut into sections. The SOFA and H&E staining were performed using Safranin O/Fast green Kit (Solarbio, China) or Hematoxylin-Eosin Staining Kit (Beyotime, China). The Osteoarthritis Research Society Internationa (OARSI) score was used to analyze the severity of cartilage degeneration (29 (link)). For IHC staining, the sections were dewaxed and treated with Antigen Retrieval Solution (Beyotime, China). The sections were incubated successfully with blocking buffer (Beyotime, China), antibodies, and diaminobenzidine (DAB) staining solution (Beyotime, China). The antibodies used in IHC staining included anti-MMP-13 (1:200; Affinity, China), anti-Collagen II (1:100; Invitrogen, USA), anti-GPX4 (1:100; Affinity, China), anti-ACSL4 (1:200; Affinity, China), anti-SLC7A11 (1:100; Affinity, China), and Goat Anti-Rabbit IgG (H+L) HRP antibody (1:500; Affinity, China).