10 cytometry analysis software

The apoptosis assay was performed on HaCaT cells using APC Annexin V Detection kit with PI (#640932, BioLegend, San Diego, CA, USA) according to the manufacturer’s instructions. Before analysis, HaCaT cells were seeded at 0.5 × 10⁶ cells/well in a 6-well plate (SPL Life Sciences, Gyeonggi-do, Republic of Korea) and incubated overnight. On the next day, the cells were treated with ORV for 24 h. The cells were then trypsinized and washed twice with Cell Staining buffer (#420201, Biolegend, San Diego, CA, USA). Annexin V binding buffer at a concentration of 1 × 10⁶ cells/mL was added to the cells that were later stained with APC annexin V and propidium iodine (PI) for 15 min at room temperature. Cells were measured for annexin V and PI intensity suing BD^® LSR II flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) and the data were analyzed using FlowJo 10 cytometry analysis software (FlowJo, Ashland, OR, USA).

For cell cycle analysis, HaCaT cells were trypsinized after treatment with ORV for 24 h. The cells were then washed with PBS buffer and fixed using 70% ethanol at −20 °C. After cell fixation, the cells were washed twice with cold PBS buffer and treated with 50 μL of 100 μg/mL RNAse to remove RNA. Finally, the cells were stained with 200 μL of Propidium Iodine (PI) and collected data of PI-stained cells using BD LSR II flow cytometer (BD Biosciences). Data were analyzed by FlowJo 10 cytometry analysis software (FlowJo, Ashland, OR, USA).