Immunofluorescence (IF) staining assay

To observe the expression of Ki67, IF staining was performed. HeLa cells were seeded onto glass coverslips in 6-well plates. Cells were incubated with lentiviruses containing shRNA-NUAK2 at 37°C for 2 days to silence NUAK2 gene expression. The cells were then incubated with si-CYFIP2 for an additional 36 h to obtain cells with low expression of NUAK2 and CYFIP2. After that, the cells were fixed with 4% paraformaldehyde (PFA; Millipore Sigma) at room temperature for 10 min, washed in PBS and blocked for 15 min in QuickBlock™ Blocking Buffer for Immunol Staining (Beyotime Biotechnology) at room temperature. After incubation with a primary antibody against anti-Ki67 (1:200; cat. no. sc-23900; Santa Cruz Biotechnology, Inc.) at 4°C overnight, cells were washed in PBS and incubated with a secondary antibody conjugated with Alexa Fluor 488 (1:1,000; cat. no. A32723; Invitrogen; Thermo Fisher Scientific, Inc.) for 1 h at room temperature. Cells were then washed in PBS, incubated for 5 min with DAPI (1 µg/ml; cat. no. D1306; Invitrogen; Thermo Fisher Scientific, Inc.), and observations were performed using a fluorescence microscope (magnification, ×100; Olympus Corporation) and were analyzed with ImageJ version 1.43 software.