Cell apoptosis assay

The Annexin V/PI assay was performed according to the manufacturer’s instructions (Thermo Fisher Scientific, Waltham, MA, USA) to analyze apoptosis. MIAPaCa-2 and Capan-1 cells were seeded into 6-well plates with 1×10⁶ cells/well and treated with cordycepin (0, 100, 200, and 400 μg/mL for both MIAPaCa-2 and Capan-1 cells). After 48 h, the cells were collected and washed with cold PBS twice, then centrifuged and resuspended at a density of 1×10⁶ cells/mL in 100 μL of binding buffer containing 5 μL of Annexin V–FITC and 1 μL of PI working solution (100 μg/mL). After incubation at room temperature for 15 min in the dark, 400 μL of binding buffer was added to each sample. The stained cells were analyzed by flow cytometry (BD Biosciences, San Diego, CA, USA) for at least 10,000 events. Cell population in different quadrants was measured by quadrant statistics.