Cell cycle analysis by flow cytometry

HepG2 cells were plated in T₇₅ flasks at a density of 1 × 10⁶cells/flask in RPMI-1640 supplemented medium. One day later, when 30% confluent, cells were treated with the indicated concentrations of I3C and sorafenib for 72 h. Then, cells were trypsinized and washed twice with phosphate buffer saline (PBS). The cellular DNA was stained using CycleTEST^TM PLUS DNA Reagent Kit (BD Biosciences, San Jose, CA) according to manufacturer’s instructions. Cell cycle analysis was performed using the Becton-Dickinson FACScan flow cytometer (BD Biosciences, San Jose, CA) and the CELLQuest software (BD Biosciences, San Jose, CA). Data from at least 10,000 events were used in each analysis. The percentage of cell distribution in G₀/G₁, G₂/M and S phases was displayed by the planimetry of the histogram.