Renilla luciferase reporter gene assay cell lysis buffer

The activity of the CTRP12 promoter was evaluated using a dual-luciferase reporter assay (30 (link)). Briefly, the CTRP12 promoter covering predicted binding sites was cloned into the pGL3 basic plasmid (Shanghai Genomeditech Co., Ltd.) with the luciferase/Renilla reporter gene. The Ov-KLF15 vector or the empty vector and luciferase/Renilla plasmids were co-transfected into H9c2 cells (1×10⁵ cells/well) using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) for 24 h at room temperature. Each well was then supplemented with 100 µl Renilla Luciferase Reporter Gene Assay Cell Lysis Buffer (Beyotime Institute of Biotechnology) and the lysed cells were centrifuged at 5,000 × g for 1 min at 4°C to collect the supernatants. Subsequently, the cells were supplemented with 20 µl lysis buffer and then with 100 µl 1X firefly and 1X Renilla luciferase reaction solutions (Wuhan Scithera Microbiological Technologies, Inc.) to detect firefly and Renilla luciferase activity, respectively. After 48 h, luciferase activity was measured using the Dual Luciferase Reporter System, purchased from Promega Corporation, according to the manufacturer's instructions. The results are presented as the ratio of luciferase to Renilla activity.

A luciferase assay was performed to detect the presence of GATA2 and miR-324-3p binding sites. Human embryonic kidney 293T cells (RRID:CVCL_0063) purchased from Shanghai Institutes for Biological Sciences (Cat# SCSP-502, Shanghai, China) were seeded into 96-well plates at a concentration of 3 × 10⁵ cells/mL (100 μL per well). When the cell density reached 70%, the cells were transfected with 0.16 μg of GATA2-WT or GATA2-MUT target plasmid in 10 μL DMEM (Invitrogen, Shanghai, China) and 5 pmol of miR-324-3p MIMIC or MIMIC NC (Hanheng, Shanghai, China), using 0.3 μL of transfection reagent (Hanheng, concentration 0.8 mg/mL). After 48 hours of incubation, the cells were lysed using Renilla Luciferase Reporter Gene Assay Cell Lysis Buffer (RG129S, Beyotime), and the relative luciferase activity was detected using a Promega Dual-Luciferase^TM Reporter (DLR^TM) Assay System (Thermo Fisher Scientific) as per the manufacturer’s instructions.