Edu incorporation assay kit

The ability of cell was detected using the Cell Counting Kit-8 (CCK-8; Dojindo, Kumamoto, Japan) according to the manufacturer’s instructions. A total of 5 × 10³cells/well were plated into 96-well plates after transfection for 24 h, and 10 μL of CCK-8 solution (Dojindo, Japan) was added on days 1, 2, and 3, respectively. Subsequently, the cells were incubated for 2 h at 37 °C, and the optical density values were measured at 450 nm using a microplate reader (Bio‑Rad Laboratories, Inc.). For the colony formation assay, transfected cells were seeded in six-well plates at 2 × 10³ per well. After 10–14 days, cell colonies were fixed with 4% paraformaldehyde, air dried, and stained with 0.05% crystal violet (Beyotime, Shanghai, China). The colonies were imaged and counted. For the 5-ethynyl-2′-deoxyuridine (EdU) incorporation assay, transfected cells were seeded into 96-well plates. The EdU incorporation assay kit (RiboBio, China) was used to evaluate cell proliferation according to the manufacturer’s instructions. Images were acquired under a fluorescent microscope at 567 nm excitation.

HCC cells treated with different doses of icaritin were cultivated with an EdU incorporation assay kit (100 ml, RiboBio, Guangzhou, China). Then, 4% paraformaldehyde was used for fixation. Following permeabilization, cell nuclei were stained by DAPI. The proliferative HCC cells were determined visually by a fluorescent microscope (Leica, Wetzlar, Germany).

The EdU incorporation assay kit (RiboBio, Guangzhou, China) was used to evaluate cell proliferation. After transfection, cells were seeded into 96-well plates at a density of 1 × 10⁴ cells/well. 24 h after seeded, 50 μM EdU was added into each well and cells were incubated for 2 h at 37 °C, fixed with 4% paraformaldehyde, then stained with DAPI. The images were acquired using a fluorescence microscope (Olympus, Tokyo, Japan).