4.7. Colony Formation Assay

Huh7 or PLC5 cells were seeded onto a 6-well plate at 1 × 10³ cells per well. After incubating for two weeks, the colonies formed were fixed in 3.7% formaldehyde solution and visualized by staining with 0.2% crystal violet. The number of colonies was counted under the microscope. Data were obtained from 3 independent experiments.4.8. Lentivirus Production and Infection

HEK293T cells were transfected with the lentivirus-based constructs along with the packaging plasmids, pCMVΔ 8.91 and pMD.G, using T-pro NTRII (T-Pro Biotechnology, New Taipei City Taiwan). Virus-containing medium was collected at 48 and 72 h post-transfection. Cells were infected with lentivirus-containing medium at a multiplicity of infection (MOI) of 10 to 25 in the presence of 10 μg/mL polybrene (Sigma-Aldrich, St. Louis, MO, USA). After 24 h, the virus-containing medium was replaced with selection medium containing 5 μg/mL puromycin (EMD Millipore). After cell growth was stable, cells were used in subsequent experiments.