Permeabilization solution

Bovine preadipocytes were seeded in six-well cell culture plates. The cells were transfected with OE-NC, OE-TORC2, siNC, or siTORC2. After 24 h, the cells were harvested, washed with 1XPBS, and re-suspended with 1× PBS containing 1 mL DNA staining solution and 10 μL permeabilization solution (Multisciences, Hangzhou, China). The suspension was vortexed for 15 s and incubated for 30 min in the dark at RT. The cell cycle was analyzed through flow cytometry (FACS CantoTM II, BD BioSciences, San Jose, CA, USA) by counting 20,000 cells.

Resuspended cells were incubated with 500 µL DNA staining solution and 10 µL permeabilization solution (MultiSciences, China) following the manufacturer's instructions. The DNA content was determined using flow cytometry (CytoFLEX S, Beckman).
Cell death was determined by propidium iodide (PI, BD, USA) staining. In brief, the cells were treated with test compounds for the indicated times, trypsinized, washed twice and resuspended in 500 µL PBS containing PI (500 ng/ml). After 20 minutes of incubation, the cell viability was detected using a flow cytometer.

For cell cycle analysis, BPH-1 and WPMY-1 cells (1 × 10⁶ cells) were harvested, washed with phosphate-buffered saline (PBS), and then centrifuged. Pellets were resuspended with 1 ml DNA staining solution, which contained 50 μg/ml propidium iodide and 0.1 mg/ml RNaseA, and 10 μl permeabilization solution (Multisciences, China). The DNA content distribution was analyzed by flow cytometry analysis (Beckman, Cat. #FC500, USA) after incubation in the dark at 37 °C for 30 min. For cell apoptosis analysis, fluorescein isothiocyanate (FITC) Annexin V Apoptosis Detection Kit I (BD Biosciences, USA) was used. BPH-1 and WPMY-1 cells (1 × 10⁶ cells) were harvested and then stained with FITC Annexin V Apoptosis Detection Kit I according to the manufacturer’s instruction.