Transfection

Lipofectamine 3000 (Invitrogen; Thermo Fisher Scientific, Inc.) was used to perform transfections following the manufacturer’s protocol. A total of 7.5 μL of Lipofectamine 3000 and final concentrations of 50 nM of siRNA for DTYMMK (5′-CGAUGUUUAACUCGGUCAACC-3′; 5′-UUGACCGAGUUAAACAUCGUU-3′) were used for each transfection in a six-well plate with 2 mL of the culture medium. Knocked-down DTYMK (target sequence: GTTTCCACCAGCTCATGAA) and negative control lentiviruses were obtained from Shanghai OBIO Technology (Shanghai, China). Huh7 and Hep3B cells were transfected with lentiviruses at a multiplicity of infection of 10. After 2 weeks of 2 µg/mL puromycin screening, Huh7 and Hep3B cells were used for subsequent experiments. Overexpressed DTYMK and negative control plasmid with a resistance of neomycin were obtained from GeneCopoeia (Guangzhou, China).