Cell cycle and apoptosis analysis kit

To measure the cell cycle of BMSCs, cell cycle was first synchronized with serum starvation before the first time of TENG stimulation and then the medium was changed with mesenchymal stem cell medium (Sciencell, No. 7501, California, USA) and started the corresponding stimulation of different groups. During this process, the culture condition between different groups was the same. Then the cell cycle was analyzed at day 7 after the seventh stimulation. Cell cycle analysis was performed using The Cell Cycle and Apoptosis Analysis Kit (C1052; Beyotime, Shanghai, China) according to the manufacturer's instructions. Analysis was then performed using flow cytometry (BD FACS Calibur; BD Biosciences, San Jose, CA).

The SLC26A9-shCOLO201 stable strain was constructed and inoculated into a 6-well plate. After 24 h, the culture was washed with PBS 3 times and fixed with 75% alcohol for 24 h. The experiment was conducted according to the instructions of The Cell Cycle and Apoptosis Analysis kit (Beyotime, Jiangsu, China). Subsequently, cell cycle analysis was performed using a flow cytometer (BD Biosciences, New Jersey, USA).

The cells were collected and fixed with ice-cold 70% ethanol overnight at 4 °C, Cell Cycle and Apoptosis Analysis Kit (Beyotime, Nantong, China) were used for cell cycle analysis. The fixed cells were stained with 0.5 mL of propidium iodide (PI) staining buffer (contains 200 mg/mL RNase A and 50 μg/mL PI) at 37 °C for 30 min in the dark. PI-stained cells were analyzed by flow cytometry (BD Biosciences, Franklin Lakes, NJ, USA).

The cells were seeded and grown in 60-mm culture plates and treated with increasing concentrations of CA or DMSO for 48 h, respectively. The cells were then trypsinized, collected, and washed using phosphate-buffered saline (FBS). After resuspending the cells, pre-cooled absolute ethanol was added to fix cells overnight. According to the manufacturer’s instruction of Cell Cycle and Apoptosis Analysis Kit (Beyotime, Jiangsu, China), 0.5 mL staining buffer, 25 µL propidium iodide staining solution (20X), and 10 µL RNase A (50X) were added to each sample, and the cell cycle distribution (5,000 events) was analyzed using flow cytometry (Beckman Coulter, United States) after incubation for 30 min.

Cell apoptosis and cell cycle were detected with the Annexin V-FITC Apoptosis Detection Kit (C1062S, Beyotime Biotechnology, Shanghai, China) and the Cell Cycle and Apoptosis Analysis Kit (C1052, Beyotime Biotechnology), performed according to the manufacturer’s instructions [17 ]. Cell apoptosis and cell cycle were measured and analyzed by a flow cytometry machine (FACS Calibur™, BD Biosciences, San Jose, CA, USA).

UA was purchased from Aladdin (Shanghai, China). Pluronic F127 was purchased from Sigma-Aldrich (St. Louis, MO, USA). C₁₈-PEI was synthesized from our previous methods.^{22 (link)} 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) were obtained from Solarbio Science & Technology (Beijing, China). Annexin V-fluoroisothio cyanate (FITC)/propidium iodide (PI) apoptosis detection kit, Cell Cycle and Apoptosis Analysis Kit and RIPA lysis buffer and blocking buffer were obtained from Beyotime (Shanghai, China). Rabbit polyclonal antibodies against caspase-3 and caspase-8 were purchased from Cell Signaling Technology (Beverly, MA, USA). Rabbit polyclonal antibodies against Fas and FasL were procured from Biosynthesis Biotechnology (Beijing, China). Rabbit polyclonal antibodies against β-actin were supplied by Proteintech (Wuhan, China). HRP-conjugated goat anti-rabbit was from Earthox (Millbrae, CA, USA). Other chemicals, if not specified, were all commercially available and used as received. The deionized water, with a resistivity of 18.2 MΩ cm⁻¹, was obtained from Milli-Q Gradient System (Millipore, Bedford, MA, USA) and used for all of the experiments.