Cell cycle analysis
HaCaT cells were seeded in six-well plates and allowed to adhere. Then the cells were serum-starved for 24 h, followed by treatment with 10%-FBS/DMEM containing various concentrations of allicin (6.4, 9.6, 12.8μg/mL) for 48 h. Later, the cells were collected by trypsinization and fixed in 70% ethanol overnight at −20°C. After being washed with PBS, HaCaT cells were resuspended and incubated with PI/RNase staining buffer according to the manufacturer’s instructions (C1052, Beyotime) for 30 min at 37°C in the dark and then subjected to flow cytometric analysis. The samples were acquired with the Attune NxT (Thermo Fisher Science), and the cell cycle distribution was further analyzed using ModFit software (Verity Software House).