Edu assay

A Cell Counting Kit 8 (CCK-8; Beyotime Biotechnology, Shanghai, China) assay was used to assess cell proliferation. Cells transfected with siRNA NC or siRNA Orc6 were transferred into 96-well plates at 3 × 10³ cells per well. After culturing for 24 h, 48 h, 72 h, 96 h, or 120 h, 100 μl serum-free DMEM/F-12 medium with 10% (v/v) CCK-8 reagent was added to the target wells. After incubation at 37°C for 2 h, the absorbance value of processed wells was detected with a microplate spectrophotometer (Eon Microplate Spectrophotometer, Biotek, Winooski, VT, USA).
An EdU assay (Beyotime Biotechnology) evaluated cell proliferation. After transfection with siRNA NC or siRNA Orc6 for 72 h, cells were seeded in 24-well plates and incubated with DMEM/F-12 medium containing 20 μmol l⁻¹ EdU for 2 h, after which cells were fixed at room temperature with 4% (w/v) paraformaldehyde at room temperature, and subsequently neutralized by adding glycine (2 mg ml⁻¹). Cells were then incubated in phosphate-buffered saline (PBS) containing 0.5% (v/v) Triton X-100 and exposed to Apollo-Fluor (RiboBio, Guangzhou, China) in the dark for 30 min. DAPI stained cell nuclei for 5 min. Percentages of EdU-positive cells were calculated by fluorescence microscopy from 500 cells.

The EdU assay (C0081s, Beyotime, China) was used to determine cell proliferation. EdU labelling, cell immobilization, Apollo staining and DNA staining were used ac-cording to the instructions, and finally, images were acquired and analysed by fluorescence microscopy.