Pgl4 luciferase reporter plasmid

The MDM2-P2 promoter region of genomic DNA was amplified by PCR. PCR products containing either SNP55T or SNP55C were inserted in tandem into a pGL4 luciferase reporter plasmid (Promega). PCR of the MDM2-P2 promoter region was performed using the following primers: forward, 5′-TATCTCGAGGTACTGGCCCGGCAGCGA-3′ and reverse, 5′-TATAAGCTTGAACACAGCTGGGAAAATGC-3′, including the XhoI and HindIII restriction site. We confirmed that the clones had different SNP55 statuses only, and that the other sequences were identical. We constructed expression vectors, pcDNA-myc-Sp1 and pcDNA-FLAG-NFκB p50, as follows. Transcription factor Sp1 and NFκB p50 were amplified from the cDNA of MCF-7 cell lines by KOD-FX (TOYOBO). During PCR, we attached a myc-tag to the N-terminus of transcription factor Sp1 using the following primers: forward, 5′-TATGAATTCGCCACCATGGAACAGAAACTGATCTCTGAAGAAGACCTGAGCGACCAAGATCACTCC-3′ and reverse, 5′-ATACTCGAGgTCAGAAGCCATTGCCACTGAT-3′ and the FLAG tag to the N-terminus of NFκB p50 using the following primers: forward, 5′-TATGAATTCGCCACCATGGACTACAAAAGACGATGACGACAAGGCAGAAGATGATCCATAT-3′ and reverse, 5′-ATACTCGAGCTAACTTCCAGTGCCCCCTCC-3′, including the EcoRI and XhoI restriction site. The amplified fragments and pcDNA3 (Invitrogen) were ligated using the EcoRI and XhoI enzymes (Takara Biotech Co) and were subcloned into pcDNA3.

For generating the NMI reporter construct, a 619 base pair region (Supplementary Information: Sequence #1) of the NMI promoter was inserted into the KpnI and NheI sites of the pGL4 luciferase reporter plasmid (Promega, Madison, WI).

Dual-Luciferase reporter assays were performed using an ICAM-1 reporter plasmid. To construct reporter plasmids, 850 bases of an ICAM-1 promoter fragment were cloned to between the XhoI and HindIII sites of the pGL4-Luciferase reporter plasmid (Promega). Briefly, 1 × 10⁴ cells were plated in each well of a 96-well plate. The next day, the reporter plasmid (100 ng) and Renilla plasmid (10 ng, Promega) were co-transfected into the cells with lipofectamine 2000 (Thermo Fisher Scientific). After 48 h, cells were washed with PBS and assayed with the Dual-Luciferase reporter assay system (Promega) according to the manufacturer’s instructions. Luciferase activity was determined using Synergy H1 Multi-Mode reader (Biotek). Quantitation of luminescent signal from reporter plasmid was normalized by quantitation of the luminescent signal from Renilla.

Binding sites between miR-373 and SIRT1 were predicted
based on a bioinformatics prediction website
(http://mirtarbase.mbc.nctu.edu.tw/php/index.php). The fragment sequences involved at the
site of action were obtained. Dual-luciferase reporter gene assay was adopted to detect
the relationship between miR-373 and SIRT1 and to
identify whether SIRT1 was indeed a direct target gene of
miR-373. According to the binding sequences of 3´- UTR of
SIRT1, both the wild type and mutation sequences were designed and
synthesized accordingly from Sangon Biotech (Shanghai, China). SIRT1
3´-UTR was cloned into pGL4 luciferase reporter plasmid (Promega, USA). Cells
were co-transfected with pGL4-SIRT1 or control pGL4 reporter plasmid and
miR-373 mimics for 48 hours by Lipofectamine™ 2000 (Invitrogen, USA).
Changes in the luciferase activity among the groups were detected using a dual-luciferase
detection kit (D0010; Beijing Solarbio Science & Technology Co. Ltd., China). The
fluorescence intensities were observed by GLomax20/20 Luminometer (E5311; Shaanxi Zhongmei
Biotechnology Co., China).

One thousand cells/well were seeded into 96‐well plates and transfected with control, UHRF1 and Nrf2 siRNA (10 nm). After 48 h, the cells were transfected with a pGL4 luciferase reporter plasmid (Promega, Madison, WI, USA) containing eight antioxidant response elements (AREs) in a final volume of 100 µl/well and luciferase assays undertaken 24 h later.