Cck 8 assay

Cell proliferation was detected by CCK8 assay (Transgen, China). For each well, 3 × 10³ cells were seeded into 96‐well plates. The cells were cultured for 24, 48, 72 and 96 h, and incubated with CCK8 at 37°C for 3 h. Then the absorbance at 450 nm was measured with a microplate reader. Cells transfected with vectors were inoculated on a 12‐well plate (2 × 10⁵ cells/well) with 70%–80% confluency. After 48 h, the cell was detected by the caspase‐3/ELISA (enzyme‐linked immunosorbent assay) (Hcusabio, China). The caspase‐3 enzyme is a marker for inflammation and apoptosis signaling, as it can regulate the destruction of DNA or cytoskeletal proteins. Each test was performed at least three times.

LCN2 chondrocytes were grown in a six‐well plate until 80% confluence was reached. Proliferation rates were evaluated using the CCK‐8 assay (TransGen Biotech). The chondrocytes (2 × 10³) were grown in 96‐well plates and incubated in a humidified 5% CO₂ atmosphere at 37 °C. After incubation for 24 h, the chondrocytes in each well were treated with 10 μl CCK‐8 and then incubated for another 2 h. Finally, the absorption was measured spectrophotometrically at 450 nm (VARIOSKAN FLASH, Agilent, Santa Clara, CA, USA). The chondrocytes were incubated with 5‐ethynyl‐2′‐deoxyuridine (EdU; Guangzhou Ribobio Co., Ltd., Guangzhou, China) for 2 h in compliance with the manufacturer's protocol. Subsequently, the chondrocytes were exposed to 200 μl 1xApollo reaction mixture for 25 min and then stained with Hoechst 33342 (5 μg/ml) for 30 min. Finally, the stained chondrocytes were examined by fluorescence microscopy and imaged. The alkaline phosphatase and von Kossa staining protocols used were as described in our previous study²⁶ and are also given in supplementary materials.

In our study, we used CCK-8 assay (TransGen, Beijing, China) and Edu incorporation assay (Ribobio, Guangzhou, China) to determine cell proliferation. The operation steps of both experiments were mainly carried out according to the instructions of the manufacturer and adjusted through the operation steps of previous studies. Additionally, we have repeated these experiments for three times.

The cell viability was detected by CCK-8 assay (TransGen Biotech, Beijing, China) following the manufacturer’s instructions. HUVECs were plated in 96-well (5 × 10³ cells/well). After IH stimulation, 10ul/well of CCK-8 was added into each well. Next, the mixture of 96-well plates was maintained at cell incubator for additional 2h. Finally, the absorbance was measured at 450nm with the use of a Multiskan GO Spectrophotometer (Thermo Fisher Scientific, USA).