Dual-Luciferase PRF Reporter Assay.

HeLa and HEK293T cells were cultured in DMEM with 10% fetal bovine serum (Thermo Fisher). PRF reporter plasmid DNAs were transfected using Lipofectamine 2000 (Thermo Fisher) according to the manufacturers’ instructions. Twenty-four hours after transfection, cells were washed once with phosphate-buffered saline (PBS), and lysed in Glo Lysis Buffer (Promega) at room temperature for 5 min. One microliter of lysate was diluted with 39 µL of PBS before being mixed with 40 µL of Dual-Glo FLuc substrate (Promega). After 10 min, FLuc activity was measured in a GloMax 20/20 luminometer (Promega). Subsequently, 40 µL of Dual-Glo Stop and Glo reagent was added to the mixture, incubated for 10 min, and measured for RLuc luminescence. The ratio between RLuc and FLuc activities was calculated as frameshift efficiency.