Cell cycle kit

The cell cycle was analyzed using the Cell Cycle Kit (BD, CA, USA). Cells were trypsinized and washed in phosphate buffered saline (PBS) for 5 min prior to collection by centrifugation at 1500 revolutions per minute (rpm). Cells were then fixed with 2 mL of prechilled 70% ethanol for 30 min at 4°C. The cells were then gently centrifuged and resuspended in a solution containing RNase A and PI. After 30 min incubations, the cells were analyzed by flow cytometry (BD Accuri C6, USA). For the apoptosis assay, the cells were trypsinized and washed with serum-containing medium. Cells were then centrifuged for 3 min at 1500 rpm and the supernatant was discarded. The cells were resuspended in 1 × Binding Buffer at 1–5 × 10⁶/mL. The cells were then stained for 10–15 min at room temperature using the Annexin V-APC/PI Apoptosis Kit (eBioscience, CA, USA) in accordance with the manufacturer's instructions. The number of apoptotic cells was analyzed using flow cytometry (BD Accuri C6, USA).

Theabrownin (>90% of purity) was purchased from Theabio Co., Ltd (Hangzhou, China; Batch number: 20151105001). Roswell Park Memorial Institute (RPMI) 1640 medium, fetal bovine serum (FBS), and 0.25% trypsin were purchased from Gibco BRL (Grand Island, NY, USA). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and dimethyl sulfoxide (DMSO) were purchased from Sigma (St. Louis, MO, USA). Annexin-V:FITC apoptosis detection kit and cell cycle kit were purchased from BD Biosciences (San Jose, CA, USA). All antibodies were purchased from Cell Signaling Technology (CST, Danvers, MA, USA). Trizol reagent and real time polymerase chain reaction (real time PCR) kit were purchased from TaKaRa (Dalian, China).

Following siRNA transfection and 8 Gy γ-ray radiation treatment, the cells were grown in an incubator with 5% CO₂ at 37°C for 24 h and then collected, fixed in 70% cold ethanol, and stored at −20°C overnight. The cell pellets were washed twice with 1 × phosphate-buffered saline and centrifuged at 1000 × g and 4°C for 10 min, following by staining with a cell cycle kit (BD Biosciences, Franklin Lakes, NJ, United States) according to the manufacturer’s instructions. The total cellular DNA content was analyzed with a flow cytometer (Beckman, Brea, CA, United States) by acquiring data for at least 10,000 events.

A dye combination of Annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) was used to stain transfected cells (BD Pharmingen, USA) after transfection. A flow cytometry analysis (BD Biosciences, CA, USA) was performed on annexin V + cells. Cells were harvested and fixed with 70% cold ethanol at − 20 °C for cell cycle analysis. Following that, cells were stained with the Cell Cycle Kit (BD Pharmingen) following the manufacturer's instructions.