35 mm plate

The 293T cells were seeded into a 35-mm plate (NEST) at a density of 2×10⁵ cells/well. The cells were grown overnight and then transfected with eIF4E small interfering RNAs (siRNAs) (sc-35284; Santa Cruz) (0 pmol, 20 pmol, 40 pmol, 60 pmol, 80 pmol, respectively) using Lipofectamine 2000 (Invitrogen Inc. USA) according to the manufacturer's instructions. Sixteen hours after transfection, cells that were transfected with eIF4E siRNAs were co-transfected with the plasmids pRHDV-luc (2 μg), peGFP (2 μg) and pIRES-Rluc (0.5 μg) using Lipofectamine 2000 (Invitrogen Inc. USA). Forty-eight hours after transfection, Western blot analysis was used to analyze the expression of eIF4E, Rluc and eGFP, in addition, The expression levels of Rluc and Fluc were evaluated using the Dual-Luciferase Reporter Assay System (Promega, USA).

RK-13 cells were seeded into a 35-mm plate (NEST) (2×10⁵ cells/well). RK-13 cells grown overnight were co-transfected with p4EBP1 (0 μg, 0.3 μg, 0.6 μg, 1.0 μg, respectively), pRHDV-luc (1 μg), peGFP (0.5 μg) and pIRES-Rluc (0.5 μg) plasmids using X-tremeGENE HP DNA transfection reagent (Roche, Switzerland). Forty-eight hours after transfection, the expression of 4EBP1, eGFP and Rluc in p4EBP1-transfected cells was analyzed by Western blotting. The expression levels of Rluc and Fluc were evaluated using the Dual-Luciferase Reporter Assay System (Promega, USA).