Luciferase Assay for Enhancer Activity

MM001 and MM047 were seeded in 24-well plates and transfected with 400 ng of pGL4.23-enhancer vector + 40 ng of pRL-TK Renilla vector (Promega) with Lipofectamine 2000 (Thermo Fisher Scientific). As positive controls, the previously published enhancers MLANA_5-I, IRF4_4-I and TYR_−9-D or ABCC3_11-I and GPR39_23-I were used for MM001 and MM047, respectively^{77 (link)}. One day after transfection, luciferase activity was measured through the Dual-Luciferase Reporter Assay System (Promega) by following the manufacturer’s protocol. Briefly, cells were lysed with 100 µl of passive lysis buffer for 15 min at 500 rpm. A total 20 µl of the lysate was transferred in duplicate in a well of an OptiPlate-96 HB (PerkinElmer) and 100 µl of luciferase assay reagent II was added in each well. Luciferase-generated luminescence was measured on a Victor X luminometer (PerkinElmer). A total of 100 µl of the Stop & Glo Reagent was added to each well and the luminescence was measured again to record Renilla activity. Luciferase activity was estimated by calculating the ratio luciferase/Renilla; this value was normalized by the ratio calculated on blank wells containing only reagents. Three biological replicates were done per condition for MM001 and two biological replicates for MM047.