Firefly luciferase reporter assay system

The cells were seeded in 6‐well plates (5 × 10⁵ cells/well) and transfected using the firefly luciferase reporter system. After 36 h, the medium was removed, and the cells were lysed with lysis buffer (Analytical Luminescence Laboratories, Mansfield, MA, USA). The level of firefly luciferase activity was measured by the Firefly Luciferase Reporter Assay System (Promega, Madison, WI, USA) according to the manufacturer's protocol.

C666-1 cells were transiently transfected with 50 ng pMirTarget luciferase reporter vector containing SOX2 3'UTR (Origene, Rockville, MD) along with 200 nM pre-miR145 miRNA precursor or pre-miR miRNA precursor control (Ambion, Austin, TX) using Lipofectamine Reagent 2000 (Invitrogen, Carlsbad, CA) mentioned above. The pMirTarget vector contains a red fluorescent protein (RFP) for monitoring the transfection efficiency and normalization. After 48 hours of transfection, the red fluorescent signal was captured under microscopy and then the cells were lysed for measurement using firefly luciferase reporter assay system (Promega, Valencia, CA) according to the manufacturer's manual. The luciferase activities were calculated and normalized to intensity of RFP expression.