Quantifying Stop Codon Readthrough and Amino Acid Misincorporation

HuH-7 sgRNA-ctrl and HuH-7 sgRNA-24 cells were seeded in 12 well plates at 30,000 cells/well. 24 hrs later cells were transfected using Lipofectamine 2000 (Invitrogen) with 0.1 μg per well of the indicated luciferase reporter construct. Cells were lysed after 24 hrs in passive lysis buffer and Rluc and Fluc activity was assessed using the Dual-luciferase Reporter Assay System (Promega) according to the manufacturer’s instructions and using a Glomax microplate luminometer (Promega). For stop codon readthrough experiments, performed in the presence of paromomycin, 1 mg/ml paromomycin (Sigma) was added to cells 8 hrs post-transfection. To measure stop codon readthrough (%), normalized Fluc activity (Fluc/Rluc) from UAG or UGA stop codon readthrough luciferase reporters was further normalized to a control construct, which does not have a stop codon between Rluc and Fluc as described (Jack et al., 2011 (link)). To measure amino acid misincorporation (%), normalized Fluc activity (Fluc/Rluc) from CGC or AAU amino acid misincorporation luciferase reporters was normalized to a control construct, which does not contain a point mutation in Fluc as described (Fujii et al., 2018 (link)). The amino acid misincorporation (%) or stop codon readthrough (%) values from the indicated number of independent experiments in HuH-7 sgRNA-ctrl and sgRNA-24 cells are shown.