Dual luciferase reporter (dlr)

Normal bladder basal stem cells were seeded at a density of 2 × 10 [4 ] cells in a 24-well format. Cells were cultured in DMEM/F12 media with B27, 20 ng/ml EGF, 20 ng/ml FGF and 4 mg/ml heparin (Life Technologies). Cells were transfected the following day using FuGENE 6 (Promega) with 2.25 μg of the TERT–luc promoter construct and 0.25 μg of pRL-TK (Promega), a control Renilla luciferase vector. 48 hours later, cells were lysed and luciferase activity was assayed with the Dual Luciferase Reporter (Promega) assay in a 96-well format according to manufacturer instructions. Experiments were performed in triplicate wells. Relative luciferase activity was calculated as the ratio of firefly to Renilla luciferase activity, to control for transfection efficiency. Control was the relative luciferase activity of cells transfected with promoterless reporter alone (pGL3-Basic).

Cells were cultured in the 48-well plates with about 60% confluence. Cell was transfected with miR-610 mimics or scramble and pGL3-TWIST1-3′UTR and PRL-TK by using Lipofectamine 2000 according to manufacturer's instructions. Renilla and firefly luciferase activitie was determined by using the dual-luciferase reporter (Promega, USA).

The bioinformatics analysis tools were applied (TargetScan (http://www.targetscan.org/vert_72/) and miRDB (http://mirdb.org/)). The luciferase pmirGLO reporter vector was performed for constructing IDO1-wild type 3′-UTR (IDO1-wt) and IDO1 mutant 3′-UTR (IDO1-mut). Cells were inoculated into a 12-hole plate at 1 × 10⁵ per hole, cultivated for one night, and cotransfected with 100 nmol of miR-760-mimics or control group plus 100 ng of pmirGLO-30-UTR. The UTR plasmid of Lipofectamine 3000 (Invitrogen) was used for 48 h. The luciferase activity was tested via a dual-luciferase reporter (Promega Corporation, Madison, WI, the United States).

A single copy of an Il5 promoter in the luciferase reporter plasmid, pGL3 Basic (Promega) and a renilla luciferase plasmid (qRL; Promega), were used. D10G4.1 cells were used for transfection by electroporation. Twenty-four hours after electroporation, cells were stimulated with PMA plus dibutyryl cyclic AMP overnight. The cell extracts were prepared and subjected to a luciferase assay using the manufacturer’s instructions for the Dual-luciferase reporter (Promega).