Luciferase reporter assays of NF- κ B and NFATc1

The transcriptional status of NF-κB and NFATc1 was gauged through a luciferase reporter assay system. In brief, RAW264.7 cells obtained from the American Type Culture Collection (Manassas, VA, United States) were transfected with either p-NF-B-TA-Luc or p-NFATc1-TA-Luc luciferase reporter constructs, which are responsive to NF-κB and Nfatc1, respectively. These transfected cells were subsequently placed onto plates (1.5×10³ cells/well) and allowed to settle overnight. The cells were pre-treated with varying concentrations of LIQ for 1 hour. Following this, the Luc-NF-κB cells that had been pre-treated were stimulated with 50 ng/mL of recombinant mouse-sRANKL for 6 h. Meanwhile, the Luc-NFATc1 cells were treated with LIQ for 24 h. Subsequent to these treatments, the cells were lysed using a luciferase reporter assay kit from Promega Corporation (Madison, WI, United States), and the luminescent signal produced by luciferase activity was quantified.