Cell Viability Assay

Cell viability was assessed with an MTT assay, following the manufacturer’s protocol (Sigma-Aldrich, Inc.). Orbital fibroblasts obtained from GO patients were seeded into 24-well culture plates (1 × 10⁵ cells/well) and treated with PCSK9 and control siRNAs (50 nM) for 10, 24, and 48 h. Thereafter, cells were washed and incubated in MTT solution (5 mg/ml) for 3 h at 37°C. Dimethyl sulfoxide (DMSO) was applied for solubilization and the absorbance of the converted dye was measured with a microplate reader (EL 340 Bio Kinetics Reader; Bio-Tek Instruments, Winooski, VT, USA) at 540 nm, with background subtraction at 630 nm.