Saci and xhoi restriction enzymes

Oligos specific to the wildtype (WT) PART1 miRNA 937-5p binding region and the mutated version of the sequence (MUT) are listed in Table S2. To make double stranded sequences for cloning, the oligos were admixed into oligo annealing buffer and heated to 90 °C for 3 min, followed by cooling to 37 °C for 15 min. The WT and MUT annealed oligos (ThermoFisher Scientific) were cloned into the multiple cloning site of the pmirGLO Dual-Luciferase miRNA Target Expression Vector (ThermoFisher Scientific, using SacI and XhoI restriction enzymes (New England Biolabs Ltd.). The confirmed vectors were co-transfected into HCC1806 cells with the pRLTK vector (Promega ThermoFisher Scientific), using TransIT-BRCA transfection reagent. 24 h later the mirVana miRNA negative control mimic or mimic-hsa-miR-937-5p (ThermoFisher Scientific) was transfected into the cells using TransIT-BRCA. The resulting firefly and renilla luciferase activity in the cells were measured 24 h later using the Dual-Glo^® Luciferase Assay System (ThermoFisher Scientifc) with a SpectraMax^® M3 Multi-Mode Microplate Reader (ThermoFisher Scientific). Binding of the mimic sequence to the luciferase reporter vector would inhibit production of luminescence.

The RelB promoter containing 898 base pairs of sequence directly upstream of the start site was cloned into the PGL3-Basic vector (Promega) using SacI and XhoI restriction enzymes (New England Biolabs). 293 cells were transfected with 500 ng of the RelB reporter construct, 50 ng pRL-TK Renilla luciferase construct (Promega) and 500 ng of either pFLAG-CMV-p65 or pCMVHA-hEZH2 (Addgene, #24230) using polyethylenimine linear (Sigma-Aldrich). The cells were harvested 24 hours later in passive lysis buffer and analyzed according to Dual-Luciferase Assay System protocol (Promega). Activity was measured using an Lmax Luminometer (Molecular Devices) and relative light units were normalized to pRL-TK Renilla luciferase light units.