293T cells were transfected with empty Circ-RLuc-IRES-Reporter vector, EMCV-IRES vector, IRES wildtype, or deletion vectors and incubated for 48 h for analyzing putative IRES activity. For Gli-luciferase reporter assay, indicated cells were seeded in six-well plates and transfected with 8xGliBS-Luc plasmid combined with pRL-TK vector (10:1 ratio) as an internal control. After 48 h, the cells were rinsed with PBS and subjected to dual luciferase assay. A dual luciferase reporter assay system (Promega, Madison, WI, USA) was used based on the manufacturer’s instructions. Firefly luciferase activity was normalized to Renilla luciferase activity for each sample. Data were from three independent assays.

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