Cell viability and apoptosis assay

To assess cell viability, AGS cells were seeded in 96-well plates and incubated with different concentrations of berberine for 24 or 48 h, respectively. Then, cell counting kit-8 reagent (Dojindo, Shanghai, China) was added to each well, followed by further incubation for 1 to 4 h. During incubation, the absorbance of cells in each well at 450 nm was measured using a microplate reader. Then AGS cells were seeded in 6-well plates and cultured overnight prior to exposure to 50 µM berberine or vehicle for 48 h. Apoptotic cells were identified by staining with annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) (Beyotime, Shanghai, China) following the instructions. The number of apoptotic cells was then immediately assessed using a FACSort flow cytometer (BD Biosciences, USA). Three biological replicates were used.