NOF and GOF strains were seeded in a 96-well plate at a density of 2500 to 5000 cells/well as previously described.7 (link) Cells were treated in triplicate with either 1X PBS (untreated), or bovine thyroid stimulating hormone (TSH, MilliporeSigma, Danvers, MA, USA) for 24 to 48 hours before addition of bromodeoxyuridine (BrdU) for 18 to 24 hours. The use of bovine TSH to stimulate the TSHR in orbital fibroblasts has been described previously.15 (link),32 (link),33 (link) Proliferation was measured with the BrdU cell proliferation assay kit (MilliporeSigma). Briefly, samples were fixed and stained with an anti-BrdU antibody followed by incubation with a corresponding horseradish peroxidase (HRP) conjugated secondary antibody. After incubation with HRP substrate, BrdU incorporation was assessed using a microplate reader (Varioskan Flash; Thermo Fisher Scientific, Waltham, MA, USA).