Luciferase activity assay kit

The protein expression plasmids for PB2, PB1, PA, NP, M1, and M1 mutants were generated by inserting their cDNA sequences into a pcDNA3.1 vector. Lipofectamine LTX was purchased from Invitrogen, and the concentration of the plasmids was 500 ng/μL. A luciferase activity assay was performed using a luciferase activity assay kit from Promega according to the manufacturer's protocol. Cell extracts were harvested 48 h posttransfection, and the luciferase activity was assayed by using the luciferase assay system. The assay was standardized against Renilla luciferase activity. All experiments were performed in triplicate.

We built pGL3-Basic-RAD18-WT and pGL3-Basic-RAD18-MUT luciferase reporter vectors (Promega, USA). The si-NC and si-E2F7 were co-transfected into 293T cells with the above two plasmids for 48 h, and the luciferase activity of each transfection group was detected by luciferase activity assay kit (Promega).

For the noninvasive assay of ectopic lesion growth in mice with endometriosis, the firefly luciferase gene was cloned into the pSMPUW-Hygro construct (Cell Biolabs, catalog number: VPK-214). Lentivirus carrying luciferase gene were generated 293 T cells by transfection with pSMPUW-Hygro containing luciferase gene and the Lenti-X high-titer lentiviral packaging system (ClonTech, catalog number: 631278). The recombinant lentivirus titer was measured using Lenti-X™ GoStix™ Plus (ClonTech, catalog number: 631280). IHEECs and IHESCs were transduced with lentiviral vectors carrying the luciferase expression cassette with TransDux MAX™ (System Bioscience, catalog number: LV860A-1). Luciferase-labeled IHEECs and IHESCs were then selected in the presence of 300 µg/ml hygromycin. The luciferase gene expression in these recombinant cells was validated using a luciferase activity assay kit (Promega, catalog number: E1980). All these recombinant cells were maintained with DMEM/F12 supplemented with 10% FBS and penicillin/streptomycin under drug selection.

NF-κB-dependent luciferase reporter plasmid (2 × NF-κB-Luc) was constructed and maintained in our lab. After 30 h of transfection, the luciferase activity assay was performed using the Luciferase Activity Assay Kit (Promega, USA). After 48 h, the assay was operated again. The relative activity of luciferase is generally calculated by normalizing the ratio of firefly/renal luciferase to negative control transfected luciferase.

The WT and mutated p53-coding regions were cloned and then inserted into the EcoRI and SpeI sites of the pGL3-luciferase vector, which was kindly provided by Dr Peifeng Li (Institute of Zoology, CAS, Beijing, China). The WT p53-coding region was mutated from ‘CACUGC’ to ‘GUGAUG’. Wild-type or mutated pGL3-p53 CDS co-transfected with negative control or the miR-142-3p duplex (Invitrogen, Shanghai, China) were transfected into HEK293T cells using Lipofectamine2000 (Invitrogen). The luciferase activity was measured using the luciferase activity assay kit (Promega). Luciferase and GFP reporter assay in zebrafish was conducted as described previously [24 (link), 58 (link)].

The wild-type CENPE (CENPE Wt) 3’UTR sequence containing a LIN28A binding site was constructed onto the pGL3-Basic vector to build the CENPE Wt reporter vector. The CENPE 3’UTR and LIN28A binding site in CENPE Wt was mutated to construct the CENPE mutation (CENPE Mut) reporter vector. The LIN28A overexpression plasmid (LIN28A) and empty plasmid (Vector) were provided by Wuhan GeneCreate Biological Engineering Co., Ltd. In K562 cells, CENPE Wt and CENPE Mut were transfected with the groups of CENPE Wt+Vector, CENPE Wt+LIN28A, CENPE Mut+Vector, and CENPE Mut+LIN28A, respectively. After 48 h of cell transfection, the change of luciferase activity was detected by luciferase activity assay kit (Promega).