The NSCLC cells were cultured until 80% confluence, and then seeded in 6-well plates in 3 ml of the RPMI medium at a cell density of approximately 105 cells/ml. The cells were subsequently treated with emodin at different dilutions (0, 25, 50, and 75 μM) in DMSO (vehicle < 0.5% volume) for 24, 48, and 72 h. Post-treatment, the cells were harvested by trypsinization and washed with PBS. The cells were then stained with 5 µL Alexa Fluor® 488 annexin V and 1 µL 100 µg/mL propidium iodide (PI) working solution per 100 µL cell sample in the dark at room temperature for 15 min, according to the manufacturer's instructions (Dead Cell Apoptosis Kit with Annexin V Alexa Fluor™ 488 & PI, Thermo Fisher Scientific, USA). After incubation the cells were kept on ice. Immediately, the apoptotic cells were determined by flow cytometry (BD FACSAria Flow Cytometer, Becton Dickinson Biosciences, USA) acquiring fluorescence emission at 530 nm and >575 nm. A small volume of cells without drug treatment were used as an unstained control.

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