Winmdi software version 2

The cells were seeded into 6-well plates (2×10⁵ cells/well), and betulinic was administrated to the cells at 0, 25, 50 and 100 µM, followed by 24 h of incubation at 37°C. DMSO was used as a control. For estimation of the DNA content, PBS was used to wash the cells, which were then fixed in ethanol at −20°C for 24 h. This was followed by re-suspension in PBS, containing 40 µg/ml PI, RNase A (0.1 mg/ml) and Triton X-100 (0.1%), for 30 min in a dark room at 37°C. Subsequently, analysis was conducted with a flow cytometer (IX-70; Olympus Corporation). The estimated percentage of cells in each phase of the cell cycle was quantified using WinMDI software version 2.0 (Informer Technologies, Inc., Los Angeles, CA, USA).

H460 cells were seeded at a density of 2×10⁵ cells/well in a 6-well plate, which were maintained for 24 h and treated with 50 µM betulinic acid for 0, 12, 24 and 48 h at 37°C in 5% CO₂ and 95% air. Thereafter, the cells from all treatment groups were collected, washed twice with PBS and re-suspended in 500 µl 3,3′-dihexyloxacarbocyanine iodide (1 µmol/l) for MMP at 37°C in a dark room for 30 min. The samples were then analyzed immediately using a fluorescence microscope (IX-70; Olympus Corporation) and a flow cytometer with WinMDI software version 2.0 (Informer Technologies, Inc.).

A431 cells were seeded at a density of 2×10⁵ cells/well in a 6-well plate and maintained for 24 h at 37°C and treated with 0, 10, 20 and 40 µM CAEfor 24 h at 37°C in 5% CO₂ and 95% air. Subsequently, cells from all samples were collected, washed twice with PBS and re-suspended in 500 µl DCFH-DA (10 µM) for ROS quantification or 3,3-dihexyloxacarbocyanine iodide (1 µmol/l) for MMP analysis at 37°C in a dark room for 30 min. The samples were then examined instantly using a flow cytometer (WinMDI software version 2.0 (Informer Technologies, Inc.).