Matrigel coated

HCC cells were seeded at a density of 1000 cells per well on a 96‐well plate and cultured for 24 hours. Then, 10 μL CCK‐8 solution (Dojindo, Japan) was added to the wells at different time points, and the cells were incubated for 2 hours. The optical density was then measured at 450 nm to determine the cell viability in each well.
For the colony‐formation assays, HCC cells were plated on a six‐well plate (1000 cells/well) and maintained in DMEM supplemented with 10% FBS for 2 weeks. The resulting colonies were then fixed with methanol and stained with 0.1% crystal violet. Photographs were captured, and the colonies were quantified using Image J software.
Cell‐migration and invasion assays were carried out on 24‐well plates with transwell inserts (8‐μm pore size; Millipore, Billerica, MA, USA). Matrigel‐coated (Millipore) inserts were used in the invasion assay. In each assay, 1 × 10⁵ HCC cells were suspended in 350 μL serum‐free DMEM in the upper chamber of the wells, and 700 μL DMEM with 10% FBS was placed in the lower chamber. The cells were incubated for 24‐48 hours, and the migrating or invading cells on the lower membrane surface were fixed in 4% paraformaldehyde and stained with crystal violet. The invading or migrating cells were counted in five randomly selected fields under a microscope (Olympus, Japan). Images were acquired at 200× magnification.

Invasion assay was done on 6-well Transwells (Millipore) with Matrigel-coated polycarbonate filters (8 μm pore size). Two × 10⁴ cells were suspended in 200 μl of RPMI medium supplemented with 0.5% FBS and seeded on the upper chamber well. The lower chamber well was filled with RPMI medium containing 10% FBS. After 48 h of incubation at 37°C, nonpenetrating cells were removed from the upper surface of the filter and penetrating cells on the lower surface of the filter were fixed with methanol and stained with Giemsa solution (Sigma). The numbers of penetrating cells were counted under a light microscope.