Firefly luciferase plasmid

Both HEK293 and Hs68 cells were grown until they reached approximately 80% confluence in 24-well plates. The cells were cultured in antibiotic-free media and co-transfected with the firefly luciferase plasmid harboring the GRE (Promega, Madison, WI, USA) and the Renilla luciferase plasmid (Promega) using Lipofectamine 3000 (ThermoFisher, Waltham, MA, USA). Following 24 h of transfection, cells were treated with the designated concentrations of dexamethasone, cAMP-elevating agents such as forskolin (Sigma-Aldrich, St. Louis, MO, USA) or IBMX (Sigma-Aldrich), or both. If required, the PKA inhibitor (H89; Sigma-Aldrich) or the extracellular signal-regulated kinase (ERK) inhibitor (PD98059; MCE, Monmouth Junction, NJ, USA) was pretreated in an incubation medium for 1 h before compound treatment. Cells were further incubated for the specified time, rinsed with ice-cold phosphate-buffered saline (PBS), and then harvested in a passive lysis buffer (Promega). For luminescence measurement, the cell lysate was mixed in sequence with the firefly luciferase reagent and transferred to a GLOMAX luminometer, followed by the addition of the Renilla luciferase reagent (Dual Luciferase Reporter assay system; Promega). The resulting luminescence was quantified with firefly luciferase activity normalized to Renilla luciferase as an internal transfection control.