Circular split nLuc reporter does not produce false positive results. (A) Schematic of the circular split nLuc reporter for screening IRES activity. False positives from a cryptic promoter or cryptic splicing cannot occur since the mRNA will not be translated into the LgBiT fused to the SmBiT. (B) Negative control IRES does not produce background signal. Luminescence from HEK293T cells transfected with plasmids encoding the circular split nLuc reporter with either the EMCV IRES, negative control IRES, or no plasmid (untransfected). (C) hPGK promoter does not produce a false positive result. Luminescence from HEK293T cells transfected with plasmids encoding the circular split nLuc reporter with either the CVB3 IRES, negative control IRES, or hPGK promoter sequence in the IRES position. (D) hPGK promoter produces a false positive signal in the bicistronic reporter. HEK293T cells transfected with plasmids encoding the bicistronic reporter with either the 42 nt random sequence negative control, an hPGK promoter, or EMCV in the IRES position. (RLU) Relative luminescence units. Data are presented as mean values +/− one SD (n = 3 biological replicates). Significance was calculated using unpaired two-tailed Student's t-test. (****) P < 0.0001, (***) P < 0.001, (**) P < 0.01, (*) P < 0.05, (n.s.) P > 0.05.
