Veritas microplate luminometer td 20 20 luminometer

The pmirGLO luciferase reporter vectors (Promega, Madison, WI) were used to construct DNA fragments from the 3’-UTR of HBP1 (Transcript: HBP1–201 ENST00000222574.4) by Pmel and Xbal (Promega) digestion according to the manufacturer’s protocol. The sequences for DNA construction and mutagenesis are listed in Supplementary Table S4. Luciferase activity was measured as described previously ^{46 (link)–48 (link)}. Briefly, cells were plated at a density of 1× 10⁴ per well into 96-well plates and then transiently co-transfected using Lipofectamine 3000 with luciferase gene reporter vectors (pmirGLO-HBP1–3’-UTR) and miR-3662 mimic (50 nmol/L) or inhibitor (100 nmol/L) according to the manufacturer’s protocol. After transient transfections for 48 hours, cells were washed twice with ice-cold PBS and were lysed in 1 x lysis buffer (Promega) for 15 min on a shaker. The luciferase activity was assessed with a Veritas Microplate Luminometer (TD-20/20 Luminometer, Turner Designs) using a Dual-Luciferase Assay System (Promega).

Cells transfected with an indicated plasmid and miRNA mimics were harvested and subjected to luciferase reporter assays using the dual luciferase assay reporter system (Promega, Madison, WI) according to the manufacturer’s instructions [30 (link)–32 (link)]. Lipofectamine 3000 was used for co-transfection of cells with miRNA mimics and a reporter plasmid containing a luciferase gene fused to the target sequence of the WT or mutant 3’-UTR of Apoptosis Inducing Factor Mitochondria Associated 2 (AIFM2). At 36 hours after transfection, cells were collected and analyzed using a Veritas Microplate Luminometer (TD-20/20 Luminometer, Turner Designs).