Assessment of cell cycle of arcyriaflavin A-treated ECSCs

The cell cycle of ECSCs following treatment with arcyriaflavin A was analyzed using flow cytometry, as previously described [5, 12]. Briefly, 72 h after arcyriaflavin A treatment (10 μM), the ECSCs were trypsinized, rinsed in phosphate-buffered saline, fixed in 70% ethanol, and then incubated for 30 min at 4 °C in the dark with a solution containing 5 μg/mL propidium iodide and 1 mg/mL RNase (Sigma-Aldrich, St. Louis, MO, USA). Flow cytometric analysis of the cell cycle was performed after propidium iodide staining using the CellFIT program (Becton-Dickinson, Franklin Lakes, NJ, USA), which analyzed the S-phase using a ModFit model.