Dual luciferase reporter gene assay kit

When the cells grew to 90% confluency, HEK‐293 cells and DB cells were seeded in 12‐well plates. HEK‐293 cells were transfected with plasmids and Lipo8000 transfection reagent. Plasmids included TOPflash reporter (Addgene, Cambridge, MA, USA), pRL‐TK, vector 1 or oeFKBP3. In another experiment, the promoter region of FKBP3 was cloned into the pGL3‐enhancer reporter vector and then co‐transfected into DB cells with FOXO3 overexpression plasmid and pRL‐TK. After 48 h, cells were lysed for the luciferase assay and the assay was performed using a dual luciferase reporter gene assay kit (KeyGEN).

AML cells (HL-60 and KG-1) were used to perform the dual-luciferase reporter assay. Potential binding sequences between CD27-AS1 and miR-224-5p, and miR-224-5p and PBX3 were predicted using Starbase and TargetScan. Cells were co-transfected with the luciferase plasmid pmirGLO (#E133A, Promega, Beijing, China) containing predicted wildtype or mutant sequences of binding targets and miR-224-5p mimic (or mimic-NC) through transfection reagent Lipofectamine 2000 (Invitrogen, NY, USA) according to manufacturer’s instruction. Relative luciferase activities (three replicates for each) were evaluated through the ratio of firefly luciferase to renilla luciferase using Dual-Luciferase Reporter Gene Assay Kit (Keygen Biotech) in accordance with the manufacturer’s protocol.

Stable cell lines with depletion of SH3RF2 were transfected with 1.5 μg pAP1-TA-luc reporter plasmids (Beyotime) and 1.5 μg pRL-TK plasmids (Promega, USA) Lipofectamine 3000 (Invitrogen, USA) following manufacturer’s instructions. One microgram pAP1-TA-luc reporter plasmids and 1 μg pRL-TK plasmids were transfected with 30 pmol RBPMS siRNA into stable cell lines with depletion of SH3RF2 by Lipofectamine 3000. The relative activity of luciferase was measured using the Dual Luciferase Reporter Gene Assay Kit (KeyGEN) as per the manufacturer’s protocols. The firefly luciferase activity was normalized to the renilla luciferase activity.

Dual luciferase reporter gene assay kit (KGAF040, KeyGEN BioTech, China) according to the manufacturer’s instruction. The NREP promoter region was inserted into the pGL3-Basic vector (BR014, Fenghui Bio, China) and co-transfected with pRL-TK, siNC, or si-HIF-1α into BC cells. HIF-1α siRNA was synthesized by General Biosystems (China). HIF-1α siRNA: 5ʹ-CAGAAAUGGCCUUGUGAAATT-3ʹ, 5ʹ-UUUCACAAGGCCAUUUCUGTT-3ʹ. After 24 h, BC cells were cultured in normoxia (N) or hypoxia (H) for another 24 h. After transfection, cells were treated with 250 μL lysis buffer. Then, 100 μL firefly luciferase detection reagent and 20 μL sample were added to each well, and the mixture was gently blown before reading. After that, 100 μL renilla luciferase detection reagent was added to each well. Luciferase activity was measured with a multifunctional microplate reader (M200Pro, TECAN, Switzerland). The relative luciferase activity was calculated by firefly/renilla.