A1202

Chondrocytes grown on the 6-well Flexcell culture plates were transferred to glass coverslips. After adhesion, chondrocytes were rinsed with PBS and fixed with 4% paraformaldehyde at room temperature for 15 min, then rinsed with PBS. Then, 1% Triton X-100 and 5% normal goat serum were added for permeabilizing for 15 min and binding nonspecific protein for 1 h, respectively. After that, chondrocytes were incubated with anti-Piezo1 (1:200, DF12083, Affinity, Liyang, China), anti-NFAT1 (1:200, A3107, Abclonal, Wuhan, China), anti-MMP3 (1:100, A1202; Abclonal, Wuhan, China), anti-MMP13 (1:200, GB11247; Servicebio, Wuhan, China), anti-COL2A1 (1:200, TA0135; Abmart, Shanghai, China), and anti-Aggrecan (1:200, TD7561; Abmart, Shanghai, China) antibodies overnight at 4 °C. After rinsing with PBST, chondrocytes were incubated with anti-mouse or anti-rabbit conjugated with CY3 and FITC for 1 h at room temperature. Next, chondrocytes were rinsed with PBST and stained with DAPI. Images were captured randomly on an Olympus BX53 fluorescence microscope. ImageJ software (version: 1.8.0) was used to quantify the mean immunofluorescent intensity.

Serial sagittal sections of embedded human and rat samples were sectioned in 6 μm and stained with HE and Safranin-O/Fast Green. OARSI scoring was used to degrade the severity of OA as reported previously [46 (link)]. For immunohistochemistry experiments, sections were incubated overnight at 4 °C with anti-MMP3 (1:200, A1202; Abclonal, Wuhan, China), anti-MMP13 (1:200, GB11247; Servicebio, Wuhan, China), anti-Aggrecan (1:400, TD7561; Abmart, Shanghai, China), and anti-COL2A1 (1:400, TA0135; Abmart, Shanghai, China). Sections were visualized using an HRP detection system and the rate of positive cells was quantified by two blinded pathologists.