Luciferase assay

Luciferase assay was performed with pMIR-REPORT™ miRNA Expression Reporter Vector System (ThermoFisher Scientific, AM5795). IB3-1/S9 cells (2.5 × 10⁵) were plated in 6-well plates 24 hours before transfection. The cells were transfected with 250 ng/ml of pMIR-Reporter, pMIR-Reporter-WT 3′-UTR, or pMIR-Reporter-mutant 3′-UTR. To control for transfection efficiency, pMIR-Reporter-β-galactosidase control vector was co-transfected. 50 nM of pre-miR-155 or pre-miR negative control was also transfected simultaneously. Transfection was performed using siPORT-NeoFX (Invitrogen). After incubating for 24 hours cells were lysed with Tropix lysis solution and luciferase activity in the cell lysates were assayed using Dual-Light Luciferase and β-Galactosidase Reporter Gene Assay System (Life Technologies, T1003). Luminescence was measured with BioTek™ Synergy™ H1 Hybrid Multi-Mode Monochromator Fluorescence Microplate Readers (BioTek Instruments, Inc.). The relative luciferase activity was normalized.