Dual luciferase analysis system

For the CD39 promoter plasmid construction, CD39 promoter region containing 1000 bp fragment was amplified by PCR from human genomic DNA using the forward and reverse primers. Then, the PCR production was cloned to pGL3.0 control plasmid between XhoI and HindIII sites. U251S and 51A GSCs grouped (PBS, SOX2siRNA) and plated in 35-mm dish 24h before transfection, followed by transfection of CD39 promoter bearing plasmid. pRL-TK was always co-transfected as the internal control. After transfection for 24h, the cells were harvested, lysed, and centrifuged for further luciferase activity analysis using Dual-Luciferase analysis system (Promega, Madison, WI, USA) according to the manufacturer’s instructions.

For the p62 promoter plasmid construction, p62 promoter region containing 1000 bp fragment was amplified by PCR from human genomic DNA using the forward and reverse primers. Then, the PCR production was cloned to pGL3.0 control plasmid between XhoI and HindIII sites. BEAS-2b cells were plated in 35-mm dish 24 h before transfection, followed by transfection of p62 promoter bearing plasmid. pRL-TK was always co-transfected as the internal control. After transfection for 24 h, BEAS-2b cells were exposed to SiNPs for another 12 h. Then, the cells were harvested, lysed and centrifuged for further luciferase activity analysis using Dual-Luciferase analysis system (Promega, Wisconsin, USA) according to the manufacturer’s instructions. The firefly luciferase activation was normalized to renilla luciferase.

HEK 293T cells were co-transfected with miR-92b-3p mimics or with a non-specific control (NC; GenePharma) and wild-type or mutated PTEN 3′-untranslated region (3′-UTR) plasmids (constructed by GenePharma) using Lipofectamine 3000 (Invitrogen, United States). Luciferase activities were measured at 48 h post-transfection using a dual-luciferase analysis system (Promega, Madison, WI, United States). Luminescence readings were acquired using a FlexStation 3 Multiscan Spectrum (Molecular Devices, Sunnyvale, CA, United States).

COS7 cells were maintained in Dulbecco's modified Eagle's medium (Thermo Fisher Scientific) with 10% fetal bovine serum (Gibco), 100 IU/mL penicillin (Sigma-Aldrich,), and 100 μg/mL streptomycin (Sigma-Aldrich), in a cell culture incubator at 37°C with an atmosphere of 95% air as well as 5% CO₂. Cells were cultured in a 12-well plate 24 h before transient transfection. Cells were transiently transfected with expression plasmids by employing the ViaFect™ Transfection Reagent (Promega). The plasmid pGL4.75 (Promega), which expresses Renilla luciferase, was cotransfected as an internal control to balance transfection efficiency, and the total amount of various plasmid DNAs per well was kept constant by supplementing the empty plasmid pcDNA3.1 where necessary. Unless otherwise indicated, 1000 ng of firefly luciferase reporter plasmid (TBX20-luc or NKX2.5-luc), 20 ng of Renilla luciferase control plasmid (pGL4.75), and 200 ng of each activator expression plasmid (wild-type SMAD1-pcDNA3.1, Tyr88∗-mutant SMAD1-pcDNA3.1 or MYOCD-pcDNA3.1, singly or in combination) were used. The luciferase activities were analyzed as described previously [21 (link)], with a dual-luciferase analysis system (Promega).