Cell cycle assay

Following incubation with different factors for 48 h, HGC-27 and SNU-216 cells were collected after trypsinization and washed twice with cold PBS. Cycle analyses were performed with the following protocol. The cells were fixed in 75% cold ethanol at −20°C overnight. Subsequently, the cells were treated with RNase A for 30 min at 37°C and stained with propidium iodide (PI) in the dark at room temperature for 30 min. Next, the cell cycle was assessed using a FACSCalibur flow cytometer (BD Biosciences), and the data were analyzed using FlowJo 10.0 software (FlowJo LLC, Ashland, OR, USA). Each experiment was repeated three times.