Sre luciferase

HEK293 cells (CRL‐1573) were obtained from the American Type Culture Collection (Manassas, VA, USA), Expi293F cells were obtained from Thermo Fisher Scientific, and B16F10‐luc‐G5 cells were obtained from Caliper Life Sciences (Hopkinton, MA, USA). The cells were cultured at 37°C with 5% CO₂. HEK293 and B16F10‐luc‐G5 cells were cultured in Dulbecco's modified Eagle's medium (Hyclone, Logan, UT, USA) with 10% fetal bovine serum and 1X Antibiotic‐Antimycotic solution (15240062, Gibco; Thermo Fisher Scientific). Expi293F cells were cultured in Expi293 Expression Medium (A1435101, Gibco; Thermo Fisher Scientific). For in vitro analyses, HEK293 cells were transfected with vectors expressing human GFRAL (OHu31183D; GenScript, Piscataway, NJ, USA), human RET (HG11997‐CF; Sino Biological), and luciferase under a serum response element (SRE) promoter (SRE‐luciferase) (E1340; Promega, Madison, WI, USA) using Lipofectamine 3000 Transfection Reagent (L3000001; Thermo Fisher Scientific).

PDGCs lentivirally infected with a dox-inducible CD97-overexpression or empty vector were transfected with luciferase signaling-reporter plasmids: cAMP response element–Luciferase (Cat# E8471, Promega), SRE-Luciferase (Cat# E1340, Promega), SRF-RE-Luciferase (Cat# E1350, Promega), and NFAT-RE-Luciferase (Cat# E8481, Promega). Twenty-four hours after transfection, cells were reseeded in black 96-well plates at a density of 75,000 cells per well with medium containing doxycycline (or dox-free medium). Forty-eight hours after transfection, cells were lysed and luciferase activity was detected using the Bright-Glo Luciferase assay system (Cat# E2650, CisBio) and a BioTek Synergy H1 microplate reader according to the manufacturer’s protocol.