Col2α

Each group of NPCs was fixed with 4 % paraformaldehyde on Petri dishes for 20 min. Following this, cells underwent three PBS washes and were permeabilized with 0.5 % Triton X-100 (Sigma-Aldrich, USA) (diluted in PBS) for 20 min. Subsequently, NPCs were blocked with 5 % Bovine Serum Albumin (BSA) for 30 min. NLRP3 (1:100, Proteintech, China), GSDMD (1:100, Proteintech, China), Col2α (1:100; Proteintech, China), and MMP13 (1:100, Proteintech, China) were then incubated with NPCs for 12 h at 4 °C. Cells were subsequently subjected to fluorescent dye incubation. This was followed by incubation with a fluorescent secondary antibody (goat anti-rabbit Alexa Fluor 488, 1:500, Beyotime, China) for 1 h at room temperature, and then with DAPI for 10 min. Images were captured using a fluorescence confocal microscope (Nikon, Tokyo, Japan). These experiments were repeated a total of five times.

Each group of the NP cells was fixed on a control dish with 4% paraformaldehyde for 20 ​min, washed three times with PBS for 3 times, and the cells were permeabilized with 0.5% Triton X-100 (diluted in PBS) for 20 ​min. The NP cells were blocked with 5% bovine serum albumin (BSA) for 30 ​min Adamts5 (1:100, Proteintech, China), MMP13 (Proteintech, China), Col2α (1:100; Proteintech, China), Aggrecan (1:100, Sigma, USA), and PGC1α (1:100, Proteintech, China) were incubated with NP cells at 4 ​°C for 12 ​h. Then, the cells were incubated with a fluorescent secondary antibody (goat anti-rabbit Alexa Fluor 488 (1:500, Beyotime, China)) at room temperature for 1 ​h, which is followed by DAPI for 10 ​min. Images were obtained using a fluorescence confocal microscope (Nikon, Tokyo, Japan). All above experiments were performed with four replicates each.