Dual luciferase reporter assay kit

Dual-luciferase reporter assay was conducted to evaluate the transcriptional activity of K-RTA [57 (link)]. WT or USP7 knockdown HEK293T cells, seeded into 24-well plates, were transfected with 50 ng of TK- renilla luciferase reporter plasmid together with 50 ng of PAN-Luc, K57-Luc or vIL-6-Luc luciferase reporter plasmid, 50ng of HA-K-RTA, and different amounts of FLAG-OTUD4 (100, 200, or 500 ng), OTUD4-C45A (100, 200, or 500 ng) or OTUD4 1-425AA (100, 200, or 500 ng). Luciferase activities were determined 24 h post-transfection using the dual-luciferase reporter assay kit (Vazyme, Nanjing, China).

For the luciferase reporter analysis, the binding site of miR-989 and its target was used as the insert sequence and clustered into the pmirGLO vector (Promega, Madison, WI, USA). XM_029863591.1 is a chitin-binding protein. Wild-type (WT) pmirGLO_XM_029863591.1_WT and mutant (Mut) pmirGLO_XM_029863591.1_Mut plasmids were designed and synthesized. In the mutant plasmid, the inserted sequence that included regions bound to the seed sequence of miR-989 was mutated by using site mutation. HEK293T cells were first cultured in an incubator at 37 °C and 5% CO₂ and then transfected with pmirGLO_XM_029863591.1 plasmids (WT or Mut) and agomir or agomir NC of miR-989 using Lipofectamine 2000 transfection reagent (Thermo Fisher Scientific) following the manufacturer’s instructions. Transfected cells were cultured in an incubator for 20 min and then transferred into new culture medium containing 10% fetal bovine serum. Luciferase activities were measured at 24 h after transfection using a Dual Luciferase Reporter Assay Kit (Vazyme). Firefly luciferase was used to normalize Renilla luciferase expression. All experiments were performed in triplicate and repeated three times.

The wild-type (WT) TGF-βRII 3ʹ-untranslated regions (3ʹ-UTR) and mutant-type (MUT) TGF-βRII (3ʹ-UTR) sequences of TGF-βRII were synthesized by GenePharma (CHN). The miR-130a-3p mimic or miR-130a-3p negative control was cotransfected into H441 cells using Lipofectamine 2000. After 48 h, luciferase activities were detected by the Dual Luciferase Reporter Assay Kit (Vazyme, CHN).

The sequences of HOTAIR or ATG10 3'UTR harboring miR-874-5p wild-type or mutant binding sites were inserted into pmirGLO vector (Promega, Madison, WI, USA) to form HOTAIR WT, HOTAIR MUT, ATG10 3'UTR WT or ATG10 3'UTR MUT reporter. Next, the constructed reporter and miR-874-5p or miR-NC were co-transfected into SK-N-SH cells. The luciferase strength was analyzed using the Dual-Luciferase Reporter Assay Kit (Vazyme).

The promoter sequence of ACSL4, PRKAA2, and SLC38A1 was synthesized and cloned to pGL3-basic vectors (Tsingke, Beijing, China), respectively. Then, 500 ng recombinant plasmids, 5 ng pRL-SV40, and miR-214 mimics or inhibitors were transfected into HEK-293FT cells. The luciferase activities were measured by Dual-Luciferase Reporter Assay Kit (Vazyme, Nanjing, China).

The miRNA 3′UTR target clone (Luc-SGMS1-3’UTR, binding site sequence; GTGTGT) was purchased from HANBIO (Shanghai, China), which contains firefly luciferase as internal control fused downstream to renilla luciferase. The HEK293 cells were co-transfected with Luc-SGMS1-3′UTR luciferase or control vector, and miR-329-3p mimic (or inhibitor) or its respective control using Lipofectamine 3000 reagent. At 24 h after transfection, renilla and firefly luciferase activities were measured using Dual Luciferase Reporter Assay Kit (cat: DL101-01, Vazyme, Nanjing, China) according to the manufacturer’s protocols. The target sites within SGMS1 3’UTR reporter were mutated to generate a mutant reporter construct (5′-CACACA-3′, bolds indicate mutations), which was used as a negative control.