Col2a1

Histological staining analyses were performed according to manufacturer's instructions. HE staining kit (G1003; Servicebio) was used for HE staining, Saffron Fast Green dye solution kit (G1053; Servicebio) for Safranin fast green staining, and Masson staining solution (G1006; Servicebio) for Masson staining.
Immunofluorescence (IF) and immunohistochemistry (IHC) analyses were conducted according with protocols.^[1] Primary antibodies for IHC included: NOTCH3 (bioss‐1812R, 1:200), α‐SMA (Proteintech, No.14395‐1‐AP, 1:3000), COL1A1 (Proteintech, No.67288‐Ig, 1:2500), COL2A1 (Proteintech, No.28459‐1‐AP, 1:800), PLVAP (bioss‐12737R, 1:50), and HEYL (Proteintech, 15679‐1‐AP, 1:200). Primary antibody for IF included fast myosin (GB112130, red light, 1:1000), slow myosin (GB112131, green light, 1:1000), NOTCH3 (bioss‐1812R, 1:200),HEYL (Proteintech, 15679‐1‐AP, 1:200), and MYOCD (bioss‐9472R, 1:200). Section area and positive area quantifications were conducted using Image J software.

Human knee articular samples and rat knee joints were prepared and fixed in 4% paraformaldehyde. Then, the samples were decalcified in 10% EDTA for 21 days and embedded in paraffin. Tissue sections (5 μm) were stained with safranin O/fast green following standard protocols to determine cartilage degradation under light microscopic examination. The Osteoarthritis Research Society International (OARSI) scoring system was used to assess joint cartilage degeneration [26 (link)]. Because both the tibial and femoral cartilages were assessed in the present study, the maximum OARSI score was 48. Three independent investigators who were blinded to the experimental groups performed the scoring. Immunohistochemistry was further performed to analyze the protein expression of KLF2, MMP13, and COL2A1 in histological sections of human knee articular samples and rat knee joints. Primary antibodies against KLF2 (Abcam), MMP13, and COL2A1 (Proteintech) were used at 1 : 100-1 : 200 dilutions and incubated overnight at 4°C. Then, the sections were incubated with a biotinylated secondary antibody. The reaction was developed using a DAB kit (BD Bioscience, Franklin Lakes, NJ, USA). The expression of KLF2 and MMP13 was evaluated by calculating the percentage of immunopositive cells. The expression of COL2A1 was evaluated by calculating the relative intensity.

Total protein separated from hADSCs using a protein extraction kit (Bio-Rad, USA), and the concentration of total protein was determined using BCA kit (Tianjin, China). Subsequently, the protein (50 μg) was separated by SDS-PAGE. Then, moved to PVDF (Millipo,USA), the membrane was incubated with blocking buffer at room temperature for 1 h. Then, incubate the first antibody GAPDH (Abcam), Aggrecan (Proteintech), SOX9 (Proteintech), COL2A1 (Proteintech), BMPR2 (Abcam) overnight, followed by secondary antibody at room temperature for 1 h. The GAPDH was used internal control. All western blot experiments were repeated at least three times.

Samples were incubated with 3% H₂O₂ for 10 min at 25°C to block endogenous peroxidase activity. After blocking with normal goat serum, primary antibodies against TNC (1:100; Proteintech, Wuhan, China), ITGB1 (1:100; Proteintech, Wuhan, China), COL2A1 (1:100; Proteintech, Wuhan, China), COL1A1 (1:100; Proteintech, Wuhan, China), p-SMAD2/3 (1:100; Cell Signaling, USA) and SMAD2/3 (1:100; Cell Signaling, USA) were added and incubated overnight at 4°C. For the immunohistochemical staining, a secondary antibody was incubated for 1 h, and then used DAB kit (ZSGB-BIO, Beijing, China) for development. After being stained with hematoxylin, the sections were sealed and photographed by a microscope. For the immunofluorescence staining, fluorescent dye-conjugated secondary antibodies FITC and CY3 (1:100; Bioss, Beijing, China) and 4′, 6-diamidino-2-phenylindole (DAPI; Beyotime Biotechnology, Shanghai, China) were used for binding and nuclear staining. Images were collected with a fluorescence microscope (ECHO, USA) and fluorescence intensity was semi-quantitatively analyzed by Image J.

Slices of the joint were prepared in the sagittal plane, with each slice measuring 4 µm in thickness for immunohistochemical analysis. Tissue expression of matrix metalloproteinases-13 (MMP-13, 1:100; proteintech, 18,165–1-AP) and collagen type II alpha 1 (COL2A1, 1:100; proteintech, 28,459–1-AP) was assessed through immunohistochemistry. The computer image analysis technique (Image-Pro Plus) was employed to analyze the percentage of positively stained cells in all the cartilage slices.

Cell lysates or milled tissues were treated with RIPA lysis buffer (Beyotime, China), and proteins were extracted. The proteins were separated by polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto PVDF membranes. The membranes were blocked with 5% skim milk. The primary antibodies used were GSDMD (Proteintech, 1 : 5000), GSDMD-N (CST, 1 : 1000), caspase-1 (Proteintech, 1 : 1000), NLRP3 (Abcam, 1 : 1000), LC-3 (CST, 1 : 1000), Beclin-1 (Boster,1 : 1000), P62 (Boster, 1 : 1000), COL2A1 (Proteintech, 1 : 6000), MMP3 (Proteintech, 1 : 4000), and GAPDH (Abcam, 1 : 10000). After incubation with horseradish peroxidase-conjugated secondary antibodies (Boster, China) and washed with Tris-buffered saline tween (TBST) buffer, the bands were visualized and detected using the enhanced chemiluminescence system. The band intensity value of proteins was calculated using ImageJ 1.52a (National Institutes of Health, USA) and normalized to GAPDH.