In vitro FXR agonist assay protocol

To directly evaluate the in vitro agonist activity towards FXR, test compounds were evaluated in CHO cells transfected with FXR responsive luciferase reporter (Nuclear Receptor & In Vitro Toxicology Solutions, Indigo Biosciences, State College, PA, USA). Cells were incubated with the test compounds for 22 h, and potency was assessed using Multi-Mode Microplate Reader (FlexStation 3, Molecular Devices Inc., San Jose, CA, USA). Efficacies are reported relative to OCA, which was set as 100% FXR activation at a concentration of 1 µM. Each compound was tested in duplicate, and the average value was reported^35,36.

The cell recovery medium (CRM, Human Farnesoid X Receptor (NR1H4, FXR reporter assay system, Indigo Biosciences, State College, PA, USA) and compound screening medium (CSM, Human Farnesoid X Receptor (NR1H4, FXR reporter assay system, Indigo Biosciences, State College, PA, USA) were removed from the freezer and thawed in a water bath at 37 °C. Test compounds were dissolved in dimethyl sulfoxide (DMSO, FUJIFILM Wako Pure Chemical Corporation, Osaka city, Osaka, Japan), and the treatment medium was prepared and diluted with CSM to achieve a final concentration of 0.2% total DMSO. The reporter cells were thawed by transferring 3.3 mL CRM at 37 °C into tubes of frozen cells. The tube containing reporter cells was recapped, and it was immediately place in a 37 °C water bath for 5 min. The reporter cells were gently inverted several times, and 100 µL cell suspension was dispensed into each well. One hundred microliters of treatment media was added into wells. Subsequently, the assay plate was kept at 37 °C, and it was incubated with 5% CO₂ for 22 h. To prepare luciferase detection reagent (LDR), the detection substrate was gently mixed with detection buffer, media contents were removed from each well, and 100 µL LDR was added to each well of the assay plate. The assay plate was allowed to rest at room temperature for 5 min, and luminescence was quantified using the Multi-Mode Microplate Reader (FlexStation 3, Molecular Devices Inc., San Jose, CA, USA)³⁶.