Id3 luminometer

The apoptosis assay was performed as previously described^{11 (link)}. To evaluate apoptosis, the luminometric Caspase-Glo 3/7 Assay System (Promega, Catalogue# G8091) was performed on cultured murine RAW 264.7 macrophages, according to the manufacturer’s protocol. Cells were seeded in 96-well plates at the density of 10,000 cells per well, grown at 37 °C and serum-starved for 24 hours. Apoptosis was induced with 1 μM STS treatment for 4 hours in the presence or absence of 10 μM atorvastatin, 4 nM SHP1i, or equal concentrations of their respective controls (DMSO, SWNT). For quantification, an iD3 luminometer (Molecular Devices) was used.

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.