A11691

Collagen, type II, alpha 1 (COL2A1), aggrecan, and MMP13 were examined by immunohistochemistry. Paraffin sections of the cartilage spheres were deparaffinized, rehydrated, pretreated with pepsin for 30 min at 37°C, and then incubated with 3% H₂O₂ in methanol solution. After flushing with PBS, the sections were blocked with BSA for 1 h at 26°C and then incubated with primary antibodies at 4°C overnight. The sections were then incubated for 15 min with the secondary antibody supplied in the HRP-Polymer Anti-Rabbit IHC Kit (KIT-5005; MaxVision, Shenzhen, China) and colored by substrates from the DAB Plus Kit (DAB-2031) for 10 min. The antibody of COL2A1 was purchased from Abcam (ab34712), the antibody of aggrecan was purchased from ABclonal (A11691; Wuhan, China), and the antibody of MMP13 was purchased from Abcam (ab39012). Knee histology images were obtained after safranine O, fast green, and Alcian blue staining. Safranine O and fast green staining were carried out for 5 min for each section to examine the cartilage in the joints. Alcian blue staining was used to stain the sections for 30 min. Images were obtained using a microscope from Zeiss and were analyzed using iViewer.

The list of regents was obtained commercially: Recombinant Rat interleukin 1 beta (IL-1β) (501-RL-010) was acquired from R&D Systems (Minneapolis, MN, USA). Rapamycin (Rapa) (S1039) was acquired from Selleck (Houston, USA) and diluted in DMSO. Antibodies against COX2 (12882; 1:1000), YAP (14074; 1:1000), P-P65 (3033; 1:1000), P-YAP (13008; 1:1000) and P65 (8242; 1:1000) were gained from Cell Signaling Technology Inc. (Beverly, USA). Antibodies against MMP13 (18165-1-AP; 1:1000), COL2A1 (28459-1-AP; 1:800), GAPDH (10494-1-AP; 1:5000), P-RIPK1 (66854-1-Ig; 1:2000) and β-actin (CL594-66009; 1:5000) were acquired from Proteintech Group (Wuhan, China). Antibodies against iNOS (A0312; 1:1000) and aggrecan (A11691; 1:1000) were acquired from Abclonal (Wuhan, China). Antibodies against P62 (GB11531-100; 1:1000) was acquired from Servicebio (Wuhan, China). Antibodies against MMP3 (BM4074; 1:500), FITC-conjugate goat anti-mouse and anti-rabbit secondary antibodies, type II collagenase and trypsin were purchased from Boster (Wuhan, China). IP lysis buffer (p0013) was provided by Beyotime (Shanghai, China).

Immunofluorescence staining was performed using primary antibodies against CD24 (1:150, ab202073, Abcam, UK), Krt19 (1:100, A19040, Abclonal, China), Col2 (1:100, A1560, Abclonal, China), Acan (1:150, A11691, Abclonal, China), MMP3 (1:100, ab52915, Abcam, UK), and Sox9 (1:100, ab185966, Abcam, UK). Fluorescence‐conjugated secondary antibodies (Invitrogen, USA) were used to visualize the fluorescent signals before the slides were stained with DAPI (Beyotime, USA). The slides were sealed using an anti‐fluorescence quenching sealer (Thermo Fisher Scientific, Massachusetts, USA). Then, fluorescence signals were captured using a fluorescence microscope (Olympus, Tokyo, Japan).