CD47 Promoter Reporter Assay

The luciferase reporter assay was performed as previously described^{11 (link)}. CD47 LightSwitch Promoter Reporter GoClones (RenSP, S710450) and Cypridina TK Control constructs (pTK-Cluc, SN0322S) were obtained from SwitchGear Genomics. 45 ng of the RenSP reporter and 5 ng of the pTK-Cluc reporter construct were transfected into mouse smooth muscle cells using Lipofectamine 3000 Transfection Reagent (Thermo Scientific, Catalogue# L3000–008) and Opti-MEM I Reduced Serum Medium (Thermo Scientific, Catalogue# 31985062). After 48 hours, media was changed to fresh medium and cells were then exposed to DMSO, 50 ng/ml TNF-α + DMSO, or 50 ng/ml TNF-α + 10 μM atorvastatin. The cell lysate and supernatant were harvested 24 hours after stimulation/treatment und dual luciferase activity was measured with the LightSwitch Luciferase Assay Kit (Active Motif, Catalogue# 32031, NC0999256) and Pierce Cypridina Luciferase Glow Assay Kit (Thermo Scientific, PI16170) using an iD3 luminometer (Molecular Devices). Relative luciferase activity (RenSP/Cypridina ratio) was quantified as the percentage change relative to the basal value obtained from control-transfected cells.