Prl sv40 renilla luciferase control vector

HeLa cells cultured in 24-well plates were transfected with indicated firefly luciferase reporter construct (SBE ×4 luc or BRE-luc) together with pRL-SV40 renilla luciferase control vector (Promega) using Lipofectamine 2000 (Thermo Fisher Scientific). Constant amount of total transfected DNA was balanced by supplying pBluescript SK vector DNA to the transfection mixture. Twenty-four hr after transfection, cells were serum-starved for 4 hr and treated with 5 ng/ml TGFβ1 for 20 hr. Cells were subsequently lysed, and firefly and renilla luciferase activities were measured using the Dual-Luciferase Reporter Assay System (Promega) according to standard protocol.

Luciferase constructs encompassed surrounding sequences of rs7175517(G/A) (NCBI: chr15:68077130-68078130,GRCh37) was cloned into the pGL3-Promoter vector (Promega, Madison, WI, United States). The luciferase constructs were synthesized by the Youbio Biological technology Co. Ltd. (Changsha, China).The constructed plasmids were sequenced to confirm the accuracy (GenScript Biotechnology Co. Nanjing, China). HEK293T cells were plated into 24-well plates in each well (7.5 × 10⁴) and cotransfected with the plasmids (100 ng/well) of interest the next day with pRL-SV40 Renilla Luciferase Control Vector (10 ng/well, Promega, Madison, WI, United States) using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, United States). After 48 h of culture, the cells were lysed, and luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI, United States). Relative luminescent signals were calculated by normalizing luciferase signals with Renilla signals. In total, 3 independent transfection experiments with triplicates for each condition were conducted.