8 μm transwell inserts

For the migration assay, 10 × 104 cells of stable cell lines (Huh7 si-NC, Huh7 si-HM13, SMMC-7721 si-NC, and SMMC-7721 si-HM13) in 250 μl of serum-free medium were inoculated in the upper chamber of 8-μm Transwell inserts (BD Biosciences, USA). Then, 500 μl of medium with 10% FBS was added to the lower chamber. We removed Huh7 si-HM13 and SMMC-7721 si-HM13 cells in the upper chamber with a cotton swab. PBS was applied to wash the migrated cells on the underside. The migrated cells were then fixed with methanol (Solarbio, China) for 10 min and stained with 10 μg/mL diamidino-phenyl-indole (DAPI, Solarbio, China) for 10 min. Positive cells in 5 random fields were photographed under a 200× inverted microscope DMI4000 B (Leica, Germany) and counted. For the invasion assay, the transwell chamber was pretreated with Matrigel, and the other experimental procedures were the same.

For the transwell migration assay, 2 × 10⁴ cells were seeded in the upper chamber of the 8-μm transwell inserts (BD Biosciences, Franklin Lakes, NJ, USA) with 100 μl of serum-free medium. Medium (500 μl) containing 10% bovine serum albumin was added to the lower chamber. After 24 h of incubation, cells in the upper chamber were carefully removed. The cells adhering to the membrane were fixed in methanol for 15 min and stained with 0.1% crystal violet (KeyGEN Biotech, Nanjing City, China) for 30 min. For the invasion assays, the upper chamber was precoated with 50 μl of Matrigel (BD Biosciences, Bedford, MD, USA) diluted 1:4 with serum-free medium, and 2 × 10⁵ cells in 100 μl of serum-free medium were seeded. The rest of the procedure was similar to that of the transwell migration assay.