Cell cycle analysis kit

After transfection, OS cells cycle analysis was determined by flow cytometry using Cell Cycle Analysis Kit (Biyuntian, China) according to the manufacturer's protocol. For apoptosis analysis, cells were treated with FITC‐Annexin V and propidium iodide (PI) in the dark, and then cells were analyzed by flow cytometry according to the manufacturer's guidelines. All experiments were performed in triplicate.

Cell cycle distribution was measured using the Cell Cycle Analysis Kit (Biyuntian, Jiangsu, China) on a flow cytometer following the manufacturer's manual. The percentages of cells in different phases were quantified.

The effects of DNA damage repair on the cell cycle of PASMCs were examined by Cell Cycle Analysis Kit (Biyuntian, Shanghai, China). PASMCs at a proper density were cultured in 6-well plates in normoxia or hypoxia and with or without transfection for 48 h. Then cells were harvested and fixed in 70% methanol overnight at 4 °C.Then cells were washed with cold PBS twice and stained in propidium iodide (PI)/RNaseA mixture. After incubation in the dark for 30 min at room temperature, cells were analysed by flow cytometry.

For CCK8 assays, MG-63-NC, MG-63-shDIAPH3, HOS-NC, and HOS-shDIAPH3 cells were seeded into 96‐well plates. After seeding for 24, 48, and 96 h, 10 µL CCK8 solution (Beyotime Institute of Biotechnology, Shanghai, China) was added to each well and incubated for 2 h. After incubation, the optical density at 450 nm was measured using a Multiskan MK3 microplate reader (Thermo Fisher Scientific). For flow cytometry analysis, MG-63-NC, MG-63-shDIAPH3, HOS-NC, and HOS-shDIAPH3 cells were seeded into 6‐well plates. After culturing for 48 h, the cells were harvested for apoptosis and cell cycle assays. Apoptosis was detected using the Annexin V-FITC Apoptosis Detection Kit (BD Biosciences, Franklin Lake, NJ, USA). The percentage of apoptotic cells was analyzed using BD FACSDiva version 8.0.1 software. Cell cycle analysis was performed using a Cell Cycle Analysis Kit (Beyotime, Shanghai, China). Cell cycle stages were analyzed using a BD LSRII flow cytometer system (BD LSRII, San Jose, CA, USA).

The experiment was performed by using a cell cycle analysis kit (Beyotime Biotechnology, shanghai, China). Briefly, cells were plated in 12-well plates at a density of 2 × 10⁵ cells /well, followed by overnight incubation. Cells were then treated with 4-OHT at a concentration of 5 μM individually or by combining with 2 mM ASA. After 72 hours’ treatment, nucleus was stained by Propidium Iodide (PI) and cell cycle was examined by flow cytometry (Beckman, USA) according to the kit's manufacturer's instructions. Data was analyzed by Flow Jo 7.6 software.

Cell cycle analysis kit (Beyotime, China) for cell cycle analysis. The cells were seeded at a density of 5 × 10³ cells per well in 96-well culture plates and transfected with miR-21 inhibitor and their negative control, respectively. Cells were washed with cold PBS after fixation, then stained with 0.5 ml of propidium iodide (PI) staining buffer at 37 °C for 30 min in the dark. The cell cycle analyses were performed on BD LSR flow cytometer. The phases of cell cycle were analyzed by WinMDI software program. The experiments were performed in triplicate.