Guavasoft software version 3

To study apoptosis induction via accumulation of the sub-G1 population, flow cytometry was carried out. LoVo and HCT116 cells were seeded at 5 × 10⁴ cells/well and allowed to grow for 24 h. Then, LoVo and HCT116 were treated with artonin E at 3, 5, 10, and 30 µg/mL and 1, 1.5, 2, 2.5, and 3 µg/mL, respectively, while the control cells were treated with DMSO. Upon treatment, both cells were harvested, washed with PBS, fixed with ice cold 70% ethanol, and then kept at 4 °C. After fixation, cells were stained according to the manufacturer’s instructions (Guava Cell Cycle^® reagent from Merck KGaA, St. Louis, MO, USA). The DNA content was observed using a Guava easyCyte™ flow cytometer and GuavaSoft™ software version 3.3 (Merck KGaA, St. Louis, MO, USA).

Sub-G1 peak analysis was performed using a flow cytometer. Cells were seeded in a 6-well plate at a density of 5 × 10⁵ cells per well and incubated for 24 h at 37 °C in a 5% CO₂ atmosphere. Next, the cells were treated with various concentrations (0, 0.6, 0.9, and 1.2 mg/mL) of stem extract. After incubation for 24 h, the cells were harvested and fixed with 70% ice-cold ethanol at 4 °C for at least 1 h. Subsequently, the cells were washed with ice-cold PBS, followed by incubation in 20 μg/mL RNase A (Sigma, St. Louis, MO, USA) and 50 µg/mL PI (Merck KGaA, St. Louis, MO, USA) with 1.5% Triton X-100 for 30 min at 37 °C in the dark. The cells were analyzed with a flow cytometer (Guava EasyCyte and GuavaSoft software version 3.3 (Merck KGaA, St. Louis, MO, USA)). The data are depicted as a histogram and the percentage of sub-G1 peaks display the apoptotic populations.