Anti acsl4

Total protein from liver tissue was extracted using RIPA lysis buffer (Solarbio, Beijing, China) containing protease inhibitor cocktail (MedChemExpress, Princeton, NJ, USA). The samples were incubated at 99°C for 5 min and separated at 115 V by SDS-PAGE for 1 h. The proteins were transferred to PVDF membranes and incubated at 200 mA for 1 h. The membrane was plugged with 5% milk powder for 1 hour and incubated overnight at 4°C with the following primary antibody: anti-FDX1 (Absin, Shanghai, China), anti-GPX4, anti-ACSL4, anti-SLC31A1 and anti-β-actin (Affinity, Cincinnati, OH, USA). HRP-conjugated goat anti-rabbit IgG was used as secondary antibodies. All bands were quantified with an automated digitizing system (ImageJ).

Total protein from cartilage tissues, FLS exosomes, and chondrocytes was isolated using radio immunoprecipitation assay (RIPA) (Beyotime, China). The protein was separated using sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) precast gel (Willget, China) and transferred to nitrocellulose membranes (Pall, USA). Then, the membranes were incubated with anti-GPX4 (1:1,000; Affinity, China), anti-ACSL4 (1:1,000; Affinity, China), anti-SLC7A11 (1:1,000; Affinity, China), anti-CD9 (1:1,000; Affinity, China), anti-CD63 (1:1,000; Affinity, China), anti-CD81 (1:1,000; Affinity, China), anti-TSG101 (1:1,000; Affinity, China), or anti-β-actin (1:5,000; Abcam, UK) primary antibodies. After incubating with a secondary antibody (1:5,000; Affinity, China), the blots were visualized using ECL Kit (Beyotime, China).

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).