Cell fit software

A549 cells were cultured in Roswell Park Memorial Institute (RPMI) (Hyclone, Logan, UT, USA) supplemented with 10% fetal bovine serum (Logan) in a humidified incubator containing 5% CO₂ in air at 37°C, and subculture was carried out with 0.25% trypsin–0.02% ethylene diamine tetraacetic acid (EDTA) (Logan). The RTHF was dissolved in phosphate-buffered saline (PBS) and adjusted to a final concentration of 10 mg/mL.
First, A549 cells (1×10⁵ cells/mL) were treated with various concentrations of RTHF (0–64 μg/mL) for different time points (24, 48, and 72 h). The A549 cells were then treated with 3 μg/mL of RTHF for 48 h. All the cell samples were then harvested, fixed in 70% ethanol (Merck KGaA, Darmstadt, Germany) and stored at −20°C for 24 h until further analysis. Next, the cells were washed twice with ice-cold PBS and then incubated with RNase and propidium iodide (PI) (Sigma-Aldrich Co., St Louis, MO, USA) for 30 min.
The PI-stained cells were excited at a wavelength of 488 nm and emitted a maximum wavelength of 617 nm. Acquisition of 10,000 events was chosen to measure the distribution of cells in the different cell cycle phases using a Coulter XL Flow Cytometer (Beckman Coulter Inc, Brea, CA, USA). The distribution of cells in the different cell cycle phases shown in the DNA histograms was analyzed using the Becton Dickinson Cell Fit Software (BD, Franklin Lakes, NJ, USA).

HepG2 and Huh-7 cells were harvested following the indicated treatment times and fixed with 75% ice-cold ethanol at 4°C for 24 h. Following washing with ice-cold PBS, cells were treated with 50 µg/ml RNase and 50 µg/ml propidium iodide (BD Biosciences; Becton, Dickinson and Company) in 500 µl binding buffer. Following incubation for 30 min, the cell samples were subjected to flow cytometry with a flow cytometer (BD Biosciences; Becton, Dickinson and Company) and data were analyzed using BD CellFIT software (BD Biosciences; Becton, Dickinson and Company). Each experiment was repeated at least 3 times.