Luciferase reporter assay

Luciferase reporter assays were conducted as previously described.23 In short, psiCheck-2 plasmids (Promega, USA) were cotransfected with different amounts of duplex RNAs (up to 75 nM) using Lipofectamine 2000 or 3000 (Invitrogen, USA). Twenty-four hours after transfection, relative activity (Renilla luciferase activity normalized to firefly luciferase) was measured using a Dual-Luciferase reporter assay system (Promega, USA) with the GloMax-multi detection system (Promega, USA) with six replicates, following the manufacturer’s protocol. IC₅₀ was calculated by performing nonlinear least-squares fitting for the sigmoid function using Scipy (scipy.optimize.curve_fit()). In cases where least-squares fitting failed to fit the function, an approximate IC₅₀ was estimated from the regression line.