Cell-based small molecule inhibitor screen

We described the generation of the MRC1 promoter luciferase construct and the establishment of a clonal THP-1 cell line stably expressing this construct previously (He and Marneros, 2014). The THP-1 MRC1 promoter luciferase reporter cells (1.0kb, clone #4) were seeded into 384-well plates at a density of 4×10⁴ cells/well by a Wellmate machine (Matrix Technologies Corp) and treated with 40 nM PMA for 24 hours and followed by the treatments of IL-4 (20 ng/ml) + 2.5 μM chemicals each for additional 3 days. The chemical libraries used were obtained from Selleckchem and included 143 natural products (library L1400–01 and L1400–02) and 1836 bioactive compounds (library L1700–1 to 21). Luciferase activity was determined using the Steady-Glo Luciferase Assay System (Promega, E2520) as per manufacturer’s protocol. All treatments in this library screen were performed in duplicate.