Shrna lentiviral infection system

FK replicon cells were infected with DUSP1 shRNA using the shRNA-lentiviral infection system (Sigma-Aldrich, St. Louis, MO). A negative control lentiviral particle (LV-cont) was also constructed. Cells in which DUSP1 expression was stably suppressed were established using shRNA-carrying lentivirus particles according to the method of Choi et al [18 (link)]. In brief, FK replicon cells were seeded at a density of 1 × 10⁵ cells per well and infected with 5 multiplicity of infection (MOI) lentiviral particles in the presence of 8 μg/mL hexadimethrine bromide (Sigma-Aldrich) overnight. Stably infected cells were selected for 2 weeks using complete medium with 500 μg/mL G418 sulfate (A.G. Scientific, San Diego, CA) and 10 μg/mL puromycin (Sigma-Aldrich). Suppression of DUSP1 expression in selected cells was confirmed by relative quantitative real-time polymerase chain reaction (PCR) and Western blot analysis.

To establish a stable Huh7 cell line depleted of ADAM17 expression, cells were infected using a short-hairpin RNA (shRNA)-lentiviral infection system (Sigma-Aldrich Co.). We constructed an ADAM17 shRNA- carrying lentiviral vector, LV-ADAM17. The shRNA negative control-lentiviral particle (LV-NC) was used as a negative control. To generate stable cells, 1 × 10⁵ Huh7 cells were plated on 12-well plates, transduced with 5 MOI lentiviral particles (using 8 μg/mL hexadimethrine bromide [(Sigma-Aldrich Co.]), and incubated in DMEM containing puromycin (5 μg/mL) at 37°C with 5% CO₂. Suppression of ADAM17 in stable cells was confirmed by RT-PCR and Western blot analyses.