Cell cycle arrest assay

PANC-1 cells were seeded in a 10 cm cell culture dish at a density of 2 × 10⁶ cells per dish and incubated for 24 h. The culture medium was then replaced with fresh medium containing different concentrations of compound KL-6 (0, 0.5, 1, 2 µM). After incubating for 24 h, the cells were trypsinized, collected, and fixed with ice-cold 95% ethanol overnight at 4 °C. Following fixation, the cells were washed with PBS and incubated with propidium iodide (PI) staining solution containing RNase A (BD Biosciences, San Jose, CA, USA) for 30 min at 37 °C in the dark. The cell cycle distribution was determined using a flow cytometer, and the data were analyzed using FlowJo software. The percentage of cells in the G0/G1, S, and G2/M phases was quantified to assess the cell cycle arrest induced by compound KL-6 (Zhang et al., 2019).