2.4. Cell Proliferation Assay

S phase cell proliferation analysis was performed using the Click-iTTM EdU Imaging Kit (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. Cells (at concentrations of 1.5 × 10³ and 2 × 10³ cells/0.32 cm²) were seeded into a 96-well plate. After three days, 2 × EdU solution was added to cells and incubated for 2.5 h at 37 °C and 5% CO₂ in humidified conditions. Cells were then fixed in 2% paraformaldehyde (PFA) for 15 min at room temperature and incubated with 40 µM Hoechst 33342 (Thermo Fisher Scientific, Waltham, MA, USA) for nuclei staining, protected from light. Following two rinses with PBS, 1X Click-iT^® EdU buffer additive was added for one hour and incubated at room temperature protected from light. Imaging and analysis of proliferating cells were performed using the ImageXpress Micro XL High-Content Screening System (Molecular Devices LLC, San Jose, CA, USA).