Cmv renilla luciferase reporter

Transient transfection and reporter gene luciferase assays were carried out as previously described. ^{43 (link),56 (link)} Briefly, 10T1/2 cells or HEK293T cells were maintained in DMEM/10% FBS with 1% penicillin and streptomycin. Then the cells were seeded in 12-well plates one day before transient transfection. Sub-confluent cells were co-transfected using Lipofectamine 3000 (ThermoFisher) with the luciferase reporter construct (in pGL3B vector, Promega) harboring SM a-actin gene promoter (nucleotide positions −2555 to +2813 containing CArG regions as reported before ^{57 (link)}), CMV-Renilla luciferase reporter as an internal control (Promega), along with or without SMYD2 and/or myocardin expression plasmids. Post-transfection 24-48 h, cell lysates were collected, and the luciferase activity was determined using the Dual-Luciferase Reporter Assay System kit (Promega) by a plate reader according to the manufacturer’s instruction. The firefly luciferase activity values in the lysates were normalized to the TK Renilla luciferase reporter activity as the internal control for transfection efficiency. The data were presented as the fold changes relative to empty vector or myocardin alone (set to 1). A minimum of 3 independent transfections was performed and all assays were replicated at least three times.

PAC1 cells were plated one day before transient transfection. Subconfluent cells were co-transfected with 50 ng of indicated SMC promoter luciferase reporter, mammalian expression plasmids and 1 ng CMV-Renilla luciferase reporter (Promega) using Lipofectamine and Plus transfection agents (Invitrogen). 24 h post transfection, the luciferase activity was determined using the Dual-Luciferase Assay System kit (Promega, Madison, WI). Relative luciferase activity was presented as the fold change after normalization with the renilla luciferase reporter activity as the internal control for transfection efficiency.