Cell Viability Assay

The MTT assay was employed to test cell viability. Following the manufacturer's protocol (Sigma-Aldrich), orbital fibroblasts isolated from GO patients and healthy non-GO donors were seeded into 24-well culture plates (1 × 10⁵ cells/well) and treated with dimethylsulfoxide (DMSO; vehicle control) and different concentrations of CHIR 99021 (1, 5, 10, and 20 µM) for 18, 24, 48, and 72 hours. Thereafter, cells were washed and incubated in 5-mg/mL MTT solution for 3 hours at 37°C. Ice-cold isopropanol was applied for solubilization, and the absorbance of the converted dye was measured with a microplate reader (EL 340 Microplate Bio Kinetics Reader; Bio-Tek Instruments, Winooski, VT, USA) at 560 nm, with background subtraction at 630 nm.