Renilla lumi luciferase reporter gene assay kit

Due to the high transfection efficiency of 293T cells, 293T cells were used and incubated on 24-well plates at 37°C. When the cells grew to about 70–80% density, the medium was replaced with serum-free MEM (Gibco, Carlsbad, CA, United States) without antibiotics, incubated overnight. The plasmid and Lipofectamine^TM 2000 (Invitrogen, Carlsbad, CA, United States) were, respectively, diluted with MEM, and incubated at room temperature for 5 min. Then they were fully mixed and placed at room temperature for 20 min. The medium in the 24-well plate was replaced with the mixture. After 4–6 h of incubation, the transfection solution was replaced with MEM containing 10% FBS. After 48 h of transfection, the cells were fully lysed. Bright-Lumi^TM II Firefly Luciferase Reporter Gene Assay Kit and Renilla-Lumi^TM Luciferase Reporter Gene Assay Kit (Beyotime, Nantong, China) and the fluorescein enzyme detection buffer of sea kidney (Beyotime, Nantong, China) were used to detect the luciferase activity with a plate reader (Promega, United States). The RLU (relative light unit) was determined by mixing 100 μl samples with 100 μl reagent solution. Cell lysate without regent was used as blank control.

The recombinant plasmids of pGL3-ADAMTS9-AS1 and pGL3-GPX4-3’UTR or their mutants were constructed in our laboratory by cloning approximately 350 bp of cDNA into the pGL3 vector (Vector Builder, Guangzhou, China). The ESCs (4.5 × 10⁴) were plated into 48-well plates and cotransfected with 70 nM miR-6516-5p or miR-cont (used as a control) with Lipofectamine 3000. Luciferase activity was detected using the Renilla-Lumi Luciferase Reporter Gene Assay Kit (Beyotime, Shanghai, China) according to the manufacturer’s protocol.