Dual luciferases reporter assay kit

According to the manufacturer’s protocol, transfections were performed by Lipofectamine 3000. A total of 3 × 10⁵ cells were seeded into each well of six-well cell culture plates 1 day before transfection, as described above. Cells were transfected with 1 µg of the luciferase reporter plus 1 ng of the Renilla luciferase reporter vector pRL-SV40, which was used as an internal control each time. The cells were washed with PBS and lysed using 1 × passive lysis buffer after 48 h. Firefly and Renilla luciferase activities were measured with a Promega Dual-Luciferases Reporter Assay Kit (Promega) according to the protocol. Each experiment was performed in triplicate, and independently repeated three times.

Total RNA was extracted from indicated cells using the Trizol kit (Omega) and reversed transcription using the cDNA synthesis kit (Takara) to obtain cDNA. Quantitative real-time PCR was then performed using SYBR Green PCR Master Mix (Takara) and CFX96 Real-Time PCR detection system (Bio-Rad). The information on primer sequences was provided in Supplementary material (Table S1). The indicated plasmids, HSF-luc, and pRL-TK were co-transfected with cells. Luciferase assays were performed using dual-luciferases reporter assay kit (Promega) according to manufacturer’s instructions.

SGC7901 or MGC803 cell is transfected with TOP-flash or FOP-flash reporter plasmid (Sigma-Aldrich) together with pRL-TK Renilla plasmid (Promega, Madison, WI, USA) with Lipofectamine 3000. 48 h post-transfection, luciferase activity is measured with a dual-luciferases reporter assay kit (Promega). Cell viability is measured using CellTiter 96 aqueous one solution cell proliferation assay (MTS) (Promega). SGC7901 or MGC803 cell is seeded in 96-well plates overnight. At various time points, 20 μl of reagent are pipetted into each well. After 2 h, the absorbance value is assessed at 490 nm under a microplate reader (BioTek Instruments, Colmar, France).

The dual-luciferase enzyme reporter assay was conducted as previously documented [17 (link)]. The wild-type (WT) docking site of miR-19a-3p on PHLDA3 and the matching mutant (Mut) were cloned into the pGL3-basic vector. The PHLDA3 WT or PHLDA3 Mut was co-transfected into 143B/U2OS cells with miR-19a-3p mimics or inhibitor or their corresponding control. After 48 h, Promega Dual-Luciferases Reporter Assay kit (Promega E1980) was performed to measure luciferase activities according to the manufacturer’s protocols.

CDK1-WT and CDK1-Mut promoters were constructed into the GL002 vector and these plasmids were transfected into A549 cells with or without NLE1 or E2F1 overexpression. The luciferase activity was measured with a Promega Dual-Luciferases Reporter Assay kit according to the manufacturer’s protocols after transfection.

The upstream sequence of BIK and different truncations were inserted into pGL3-based luciferase reporter plasmids. The followed primers were used for PCR. UP: 5- GCGGTACC ACCCAACAGGTAGCAA-3, DN:5- GCCTCGAGGGCCCGGCTGCCGGCGC-3 for P1; UP: 5- GCGGTACC ACCCAACAGGTAGCAA-3, DN: 5- GCCTCGAGACAAATATGAAAACCGAGG-3 for P2; UP: 5- GCGGTACC GAAATAGGCTTTAAAACA-3, DN: 5-GCCTCGAGGGCCCGGCTGCCGGCGC-3 for P3. The sequence of KLF4 3’UTR was cloned into pSICHECK2 vector. The followed primers were used for PCR: UP: 5-GCCTCGAG ATCCCAGACAGTGGATAT-3. DN: 5-GCGCGGCCGC TTCAGATAAAATATTAT-3. The plasmids were transfected into osteosarcoma cells and after transfection, luciferase activity was measured in a 1.5-ml Eppendorf tube with the Promega Dual-Luciferases Reporter Assay kit (Promega E1980) according to the manufacturer’s protocols. Relative Renilla luciferase activity was normalized to firefly luciferase activity. The assay was performed as previously described [22 (link), 23 (link)].