RORγt-GAL4 luciferase assays.

The RORγt-GAL4 fusion protein was a gift from the lab of Dan R. Littman at New York University Langone Health. This protein was used to drive luciferase activity from the PGL4.31[luc2p/GAL4UAS/Hygro] (luc2p) vector (Promega, catalog no. C935A). The pRL-CMV vector (Promega, catalog no. E226A) was used as the internal control Renilla luciferase reporter. Cells were plated according to manufacturer instructions for transfection reagents. Lipofectamine 2000 transfection reagent (Thermo Fisher Scientific, catalog no. 11668019) was used for human lung adenocarcinoma H522 cells and TransIT-X2 transfection reagent (Mirus Bio, catalog no. MIR6000) was used for human Jurkat T cells. At 48 hours after plating, the H522 cells were washed twice with ice-cold PBS in the plate, and Jurkat cells were collected into Eppendorf tubes and spun down before PBS washes and luciferase activity measurements using the Dual Luciferase Reporter Assay (Promega, catalog no. E1910). Cells were lysed in 1 part Passive Lysis Buffer (Promega) made from a 1:5 dilution of 5× Passive Lysis Buffer. Samples were allowed to rock at room temperature for 30 minutes before a quick centrifugation step at 12,000 g and 4°C for 2 minutes. Samples were plated in a solid white polystyrene 96-well plate (Corning, catalog no. 353296) at 20 μL/well. LARII and Stop & Glo Reagent were prepared according to manufacturer’s instructions. Plates were read in a GloMax 96 Luminometer (Promega, catalog no. E4861).