Beyoclick edu detection kit

EdU incorporation assay was performed according to the manufacturer's instructions (BeyoClick™ EdU Detection Kit; C0075S, Beyotime Biotechnology). All reagents used in the assay were provided by the manufacturer. Both Huh7 and Hep3B cells were seeded on coverslips in 24-well plates at 4 × 10⁴ cells per well and treated with EA or untreated control. After 24 h, both treated and untreated cells were incubated with EdU (20 μM) for 1 h. Next, cells were washed with phosphate-buffered saline (PBS) three times and fixed with fixation buffer for 15 min at room temperature, followed by penetration for 15 min at room temperature. Cells received an additional three rounds of PBS washes followed by incubation with click reaction buffer for 30 min at 37 °C and washed again with PBS. Nuclei were stained with Hoechst at 1:1000 for 10 min at room temperature and then washed with PBS. Finally, cell-seeded and stained coverslips were mounted and imaged using a Leica fluorescence microscope. Three random fields per slice were imaged to quantify the EdU incorporation rate, which was represented by the ratio of EdU-positive cell number to Hoechst-positive cell number. This experiment was repeated in triplicate.