Ov nc

The small interfering (si)RNA-negative control (NC), siRNA-AKIP1-1/2, empty vector (Ov-NC) and Ov-Rac1 constructs were purchased from Guangzhou Ribo Biotechnology Co., Ltd. (https://www.ribobio.com/). ccRCC cells were seeded into 6-well plates at a density of 1x10⁵ cells/well in complete medium and were cultured at 37˚C. When cells reached 70% confluency they were transfected with siRNA-NC (siB06525141922-1-5; 60 nM), siRNA-AKIP1-1 (siG1281084018-1-5; 60 nM), siRNA-AKIP1-2 (siG1281084034-1-5; 60 nM), Ov-NC and Ov-Rac1 at 37˚C using Lipofectamine^® 3000 (Invitrogen; Thermo Fisher Scientific, Inc.) for 24 h. The cells were collected for further experimentation following 48 h of transfection.

The chondrogenic cell line, ATDC5, was purchased from RIKEN BioResource Center and cultured in DMEM (Gibco; Thermo Fisher Scientific, Inc.) containing 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) at 37˚C in a humidified atmosphere with 5% CO₂. The cells were stimulated with IL-1β (10 ng/ml) for 24 h and then 5, 10 and 20 mg/ml KT were used to treat these IL-1β-induced ATDC5 cells at 37˚C for 24 h; IL-1β-induced ATDC5 cells without KT as used as the control.
With the aim of overexpressing COX-2 expression in ATDC5 cells, the pcDNA3.1 vector containing full-length COX-2 (OV-COX-2) and empty vector (OV-NC) were all designed and synthesized by Thermo Fisher Scientific, Inc. In addition, the cells that were not transfected with the plasmid were used as the control group. The transfection of ATDC5 cells was performed with Lipofectamine^® 2000 transfection reagent (Invitrogen; Thermo Fisher Scientific, Inc.) at a concentration of 50 ng/ml. Following transfection for 48 h at 37˚C, the transfection efficiency was detected by reverse transcription-quantitative PCR (RT-qPCR) 48 h post-transfection.

For overexpression of REC8 and CDC20, pcDNA3.1 vector containing full-length REC8 (OV-REC8) and CDC20 (OV-CDC20) and corresponding empty negative control vectors (OV-NC) were designed and synthesized by Thermo Fisher Scientific, Inc. Untransfected cells were used as the control. Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) was used to transfect plasmids into cells (1×10⁵ cells/well) at room temperature at 50 ng/ml according to the manufacturer's instructions. After 12 h, the medium was replaced with fresh DMEM and cells were routinely cultured for 72 h at 37°C.

Two small interfering (si)RNAs specific to SPTBN2 (siRNA-SPTBN2-1, 5'-ACGTCAATGTACACAACTTCACC-3'; and siRNA-SPTBN2-2, 5'-ACCATTACTTCTCCAAGATGAAG-3'), a pcDNA3.1 expression vector carrying ITGB4 (Ov-ITGB4) as well as their corresponding negative controls (NC) si-NC (5'-AAGACAUUGUGUGUCCGCCTT-3') and Ov-NC (empty vector) were purchased from GeneChem, Inc. Using Lipofectamine^® 2000 (Thermo Fisher Scientific, Inc.), 100 nM siRNA-SPTBN2-1/2, siRNA-NC and 4 µg Ov-ITGB4 and Ov-NC were transfected into A2780 cells at 37˚C for 24 h, according to the manufacturer's protocol. After 48 h, cells were washed with PBS and incubated in DMEM and reverse transcription-quantitative PCR (RT-qPCR) and western blotting were performed to test transfection efficacy.