Dual luciferase assays and miRNA mimics

The miR-5119 mimic (Qiagen) and control miRNA (Qiagen, #1027281) were transfected into 293T cells in combination with the PD-L1 3′ UTR pmirGLO dual luciferase reporter plasmid. The miR-5119 mimic sequence was 5′-CAUCUCAUCCUGGGGCUGG-3′. 293T cells were transfected using Endofectin according to the manufacturer’s protocol. 293T cells (1 × 10⁵) were transfected with miRNA (1 μg/ml), plasmid (1 μg/ml), and Endofectin (2 μl/ml) wells of a 96 well plate. After co-transfection a dual luciferase reporter assay was performed using a Lumat LB 9507 luminometer (Berthold Technologies, Oak Ridge, TN). Both firefly luciferase and renilla luciferase activity were detected using a dual-Luciferase Reporter Assay System (Promega, US) according to manufacturer’s introduction.