Rnase a

Cells at a density of 3 × 10⁵ cells/well were seeded into a 6-cm Petri dish and incubated with completed medium at 37 °C for 24 h. Subsequently, the cells were treated with Huaier extract for 48 h, and the total cells were collected. The cells were fixed with 75% cold ethanol (1 mL of PBS and 3 mL of absolute ethanol) at −20 °C overnight. Then, the DNA of cells was stained with 200 of μL RNase A (1 mg/mL) and 500 μL of propidium iodide (PI, 100 μg/mL) (Liankebio, Zhejiang, China) for 30 min at room temperature in the dark, and they were analysed using a FACScan flow cytometer. The data were analysed using ModFitLT software, version 2.0 (Becton Dickinson, Franklin Lakes, NJ, USA).

3 × 10⁵ cells were seeded into each well on a 6 cm Petri dish the day before. The cells were collected after incubated with Huaier n-butanol extract for 48 h. The cells were fixed with 75% cold ethanol at −20 °C overnight. Then, the cells were stained with 200 of μL RNase A (1 mg/mL) and 500 μL of propidium iodide (PI, 100 μg/mL) (Liankebio, Zhejiang, China) for 30 min at room temperature in the dark, and they were analyzed using a FACScan flow cytometer. The data were analysed using ModFitLT sofware, version 2.0 (Becton Dickinson, Franklin Lakes, NJ, USA).