Aggrecan

Samples were washed with PBS three times and then blocked with 5% BSA solution for 30 min, followed by incubation with the primary antibodies diluted in 5% BSA solution overnight at 4 °C: eNOS (ab5589, Abcam), alpha smooth muscle actin antibody (αSMA, ab7817, Abcam), CD68 (MCA341GA, BioRad), Cyclin D1 (A19038, ABclonal), Aggrecan (13880-1-AP, Proteintech), and CD206 (ab64693, Abcam). After that, samples were washed with PBS three times and incubated with goat anti-rabbit secondary antibody or goat anti-mouse antibody for 1 h at room temperature. Nuclei were counterstained with DAPI. Tissue sections without primary antibody incubation were used as negative controls. The stained samples were observed with an inverted microscope (IX73, Olympus, Japan). Five different tissue sections (n = 5) from five different rats for each group were quantified. Specifically, for CD68⁺ and CD206⁺ cell quantification, two different tissue sections from the same sample were stained with CD68 and CD206 antibodies, respectively. The numbers of the CD68⁺ and CD206⁺ cells and the ratio of CD206⁺ cells to CD68⁺ were then quantified. Five sets of tissues sections (n = 5) from five different rats for each group were quantified.

The collected NP tissues was ground to powder in liquid nitrogen. The cells at 24 h after treated with mechanical stress or above tissues powder were placed in RIPA lysis buffer (Beyotime, China) supplemented with 1 mM PMSF (Beyotime, China) on ice for 30 min. The collected liquid was centrifuged at 12,000 rpm for 15 min at 4 ℃. The protein concentration was detected with a BCA protein assay kit (PC0020, Solarbio). The protein samples from each group were separated in 8%, 10%, or 12% SDS–polyacrylamide gels (SDS-PAGE) and then transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, USA). After blocking with QuickBlockTM Blocking Buffer (Beyotime, China) for 20 min at room temperature, the membranes were incubated with rabbit primary antibodies against ACSL4 (1:2000, Proteintech), Bip (1:2000, Proteintech), Piezo1 (1:1000, Proteintech), Calnexin (1:5000, Proteintech), GPX4 (1:1000, Proteintech), ATF6 (1:2000, Proteintech), PERK (1:1000, Proteintech), Aggrecan (1:1000, Proteintech), Col-2 (1:1000, Novus), ADAMTS-5 (1:1000, Abcam), MMP-13 (1:1000, Proteintech), SelK (1:500, Proteintech), GAPDH (1:5000, Proteintech) overnight at 4 ℃. Then, the membranes were incubated for 90 min at room temperature with secondary antibody. The bands were visualized using an Amersham Imager 600, and the density was quantified using ImageJ software.