Cell cycle assay kit

CCK-8 and cell-cycle-assay kits(KeyGEN, Jiangsu, China) were used to assess the proliferation of the cells. For CCK-8, a 96-well plate was used with approximately 8000 cells per well. After the cells were cultured overnight, the medium was replaced with H-DMEM with 1%(v/v) FBS and tFNAs at different concentrations. For the control group, the cell medium contained no tFNAs. Cell proliferation was examined after 24 h of treatment with CCK-8 solution. A flow-cytometry assay was used to examine the cell cycle. After 24 h of treatment, samples were collected with 0.25% (w/v) trypsin-EDTA solution and fixed with 70% ethanol at 4 °C for 12 h. After rewashing with PBS, the samples were incubated with 50 μL of RNase at 37 °C for 30 min. Next, 450 μL of propidium iodide (PI) solution was added to samples for 30 min at 4 °C in the dark. The percentages of cells in the G0-G1, S, and G2-M stages were then measured using a Millipore Guava easyCyte HT (Burladingen, Germany). The changes in the cell-cycle distribution were investigated using FlowJo software.

Flow cytometry was conducted to detect the cell cycle distribution and apoptotic rate. The Annexin V−FITC/PI cell apoptosis detection kit and cell cycle assay kits were bought from NanJing KeyGen Biotech Co., Ltd. (cat. no. KGA108). Cells were digested with 0.25% pancreatin (MedChemExpress; cat. no. HY−B2118) without EDTA. Cell apoptosis was assessed using the AnnexinV−FITC/PI cell apoptosis detection kit according to the manufacturer’s protocol. Briefly, cells were re−suspended in 500 µl binding buffer mixed with 5 µl AnnexinV−FITC, then mixed with 5 µl PI and incubated at room temperature in the dark for 15 min. The cell cycle was assessed using the cell cycle detection kit according to the manufacturer’s protocol. Cells were washed with PBS, centri−fuged with 350 x g for 5 min at 4°C, fixed with pre−cooled 70% ethanol at 4°C for 1−2 h, washed for a second time and the cell suspension was stained at 37°C for 15 min with 1 ml PI/Triton X−100 (20 µg PI/0.1% Triton X−100) containing 0.2 mg RNase.

PD 0332991(CDK4/6 inhibitor; #HY-50767, CAS Number: 571190-30-2) was purchased from MedChemExpress LLC (Shanghai, China). DDP (cisplatin; #BP809, CAS Number: 15663-27-1), N-Acetyl-L-cysteine (NAC; #A7250, CAS Number: 616-91-1), 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA; #287810, CAS Number: 4091-99-0) and Dimethyl sulfoxide (DMSO; #D2650, CAS Number: 67-68-5) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Cell cycle assay kits (#KGA512) were purchased from KeyGEN BioTECH (Jiangsu, China). Superoxide Dismutase (SOD; # S8410, CAS Number: 9054-89-1) was purchased from Beijing Solarbio Science & Technology Co. (Beijing, China). Catalase (#MB3116, CAS Number: 9001-05-2) and Glutathione (GSH; # 571190-30-2, CAS Number: 70-18-8) were purchased from Dalian Melun Biotechnology Co. (Dalian, China). MK2206 (Akt inhibitor; # S1078, CAS Number: 1032350-13-2) was purchased from Selleck Chemicals (Shanghai, China). A549-myr-Akt cells were a kind gift from Dr Junchao Cai, Sun Yat-sen University Zhongshan School of Medicine [36 (link)].