Cell cycle kit

For cell cycle analysis, cell-cycle distribution was determined with the cell cycle kit (#KGA512, Keygen, China) by flow cytometry (CytoFlex, Beckman, USA). Briefly, the cells were seeded in the 6-well plates, and different concentrations (0.5, 1.0, 2.0, 3.0, and 4.0 μM) of NMN were added. After culturing for 48 h, cells were digested with trypsine. The single cells were fixed with 75% ethanol for 2 h at 4°C. After fixing, the cells were washed thrice with PBS and centrifuged at 500 × g. The PI/RNase solution was added to the cells, stained in the dark at room temperature for 30 min, and detected by flow cytometry at the FL3 (Ex = 488 nm, Em ≥ 630 nm) channel. The treatments were assessed in triplicate, and the experiment was independently repeated three times. After flow cytometry, data were analyzed with ModfitLT software.

The cell cycle was analyzed by flow cytometry. Briefly, primary fibroblast cells were treated under different conditions was used for analysis. After treatment, cells were collected and washed twice with ice-cold 1 × PBS, suspended in 700μl of pre-cooled 80% alcohol, and fixed the cells at 4°C for 4 hours. The cells were then stained with the Cell cycle kit (Keygen, Nanjing, China). The cells were washed and analyzed by flow cytometry using cytoFlex (Beckman coulter).

HCT116 and LOVO cells were treated with 0, 50, 100, and 200 μg/ml of ASP for 24 h. The collected cells (1 × 10⁶) were fixed in cold ethanol and stored overnight at 4°C. The next day, cells were washed twice with cold PBS; then 100 μl of RNase A (25 μg/ml) and 400 μl of propidium iodide (PI, 50 μg/ml) were added to each sample according to the instructions of the cell cycle kit (KeyGEN, Nanjing, China) and incubated for 40 min in the dark. The cells were analyzed using a flow cytometer (BD Biosciences, San Jose, CA, United States) and FlowJo 10.0 software (FlowJo, Ashland, OR, United States).

Human Burkitt lymphoma cell lines Raji and Ramos were donated by Jiangsu Key Laboratory of the Noncoding RNA Foundation and Clinical Transformation of Yangzhou University. The RPMI 1640 cell culture medium (GIBCO) and fetal bovine serum (FBS) (Hyclone Laboratories Inc.) were purchased from Abbkine Scientific (China). CCK8 was also obtained from Abbkine Scientific (China). A hematoxylin-eosin staining kit was purchased from Guangdong Baso (China). A cell cycle kit was purchased from KeyGEN BioTECH (China). The EdU staining kit, JC-1 stain kit, and Annexin V-PI kit were purchased from Shanghai Beyotime (China). Monoclonal antibodies against Bcl-2, BAX, Cleaved-caspase-3, and Bcl-xL were purchased from Cell Signaling Technology (United States). The β-actin antibody was obtained from Sigma-Aldrich (United States).