For CCK‐8 assay, Huh7 cells were seeded into six‐well plates and transfected with the indicated plasmid when they reached 80% confluence. Transfected cells were digested with 0.25% trypsin, and 3000 cells were seeded into each well of a 96‐well plate. The WST‐1 Cell Proliferation and Cytotoxicity Kit (Beyotime) was used to measure proliferation according to the manufacturer's protocol. In brief, 10% WST‐1 solution was added to cell culture medium in each well, and the optical density at 450 nm was measured at 0, 24, 48, and 72 hours. Six duplicate wells for each group were measured.
For key Fluor488‐EdU staining assay, briefly, Huh7 cells were transfected with individual plasmids and incubated with 1× EdU working solution for 30 minutes, 4% PFA was used to fix cells, and Hoechst 33342 staining solution was incubated with cells for 15 minutes. At least four random fields of each well were obtained at 480 nm (green) and 350 nm (blue). Green represented proliferating cells and blue represented cell nucleus. Ratio of EdU staining = (proliferating cells/whole cells) × 100.
For key Fluor488‐EdU staining assay, briefly, Huh7 cells were transfected with individual plasmids and incubated with 1× EdU working solution for 30 minutes, 4% PFA was used to fix cells, and Hoechst 33342 staining solution was incubated with cells for 15 minutes. At least four random fields of each well were obtained at 480 nm (green) and 350 nm (blue). Green represented proliferating cells and blue represented cell nucleus. Ratio of EdU staining = (proliferating cells/whole cells) × 100.