5 ethynyl 2 deoxyuridine edu assay kit

The 5‐ethynyl‐2′‐deoxyuridine (EdU) Assay Kit (Beyotime, C0078S) was used to conduct the EdU assay in accordance with the specifications of the kit manufacturer. Briefly, the cells disposed of by ritanserin for 48 h or transfected with scramble and targeting siRNAs were cultured in a 96‐well plate and collected the next day. Next, the cells were incubated in a 37°C incubator for ~1 h and 30 min after the addition of 1000× dilution of the EdU working solution. Next, we fixed the cells with 4% paraformaldehyde for 15 min. Subsequently, the cells were incubated in the permeable solution containing 0.3% Triton X‐100 in PBS. The cells were incubated with the Click Additive Solution for 30 min and with the Hoechst 3342 reagent for 10 min, respectively. Finally, the relevant pictures were captured at 20× magnification under a fluorescence microscope.

Cell viability was assessed using a Cell Counting Kit-8 (CCK-8; Beyotime Institute of Biotechnology) and a 5-ethynyl-2′-deoxyuridine (EdU) assay kit (Beyotime Institute of Biotechnology). For the CCK-8 assay, transfected cells were seeded into a 96-well plate at a density of 5,000 cells/well. After incubation in a 37°C incubator for 24 h, CCK-8 reagent was added into each well and the cells were incubated for another 2 h at 37°C. The absorbance values were then measured at 450 nm. For EdU assays, cells were seeded into 96-well plates (5×10³ cells/well) and incubated for 24 h at 37°C, then fixed in 4% paraformaldehyde at room temperature for 30 min and stained with EdU reagent for 2 h at 37°C, followed by DAPI staining at room temperature for 20 min. Images were captured under a fluorescence microscope (Leica Microsystems GmbH).