8 cck 8 assay

The proliferation of HKFs was assessed using a cell counting kit-8 (CCK-8) assay (Dojindo, Kumamoto, Japan) according to the manufacturer protocol (Wu et al., 2020 (link)). Briefly, HKFs were plated in a 96-well plate at a density of 3 × 10³/well and incubated to 40% confluence. After transfection, 10 μL CCK-8 reagent was directly added to each well of a 96-well plate at the specified time (0 h, 24 h, 48 h, 72 h, and 96 h), and then incubated for 2 h in a dark environment. Finally, a microplate reader (BioTek Instruments, United States) was applied to measure the optional density (OD) at a wavelength of 450 nm.
The proliferation of HKFs was also measured with an EdU DNA cell proliferation kit (RiboBio, Wuhan, China). After transfection, the HKFs were incubated with a medium containing 50 μM EdU for 2 h. The HKFs were fixed with 4% paraformaldehyde for 30 min, and then the excess formaldehyde was neutralized with a 2 mg/mL glycine solution. Subsequently, the HKFs were, respectively, stained in Apollo reaction cocktail and Hoechst staining solution and then incubated for 30 min in the dark. A fluorescence microscope (IX35, Olympus, Japan) was used for capturing randomly selected areas to observe the ratio of proliferating cells (EdU positive) to the total number of cells (DAPI positive) (Bieg et al., 2019 (link)). Samples were prepared in triplicate.

Cell counting kit-8 (CCK-8) assay (Dojindo) was used to measure cell proliferation following the manufacturer’s guidelines. Briefly, cells were seeded in 96-well plates (1000 cells per well) and cultured for the indicated days. To measure cell number, 10 µl CCK-8 was added into each well, followed by continuous incubation for 4 h. The optical density value of cells in each well was measured at the wavelength of 450 nm on a microplate reader. The assay was carried out in triplicates and repeated three times.