2.5. Cell Viability (MTT Assay)

Cell viability was measured using an MTT assay. MDA-MB-231 cells were seeded in 12-well plates and incubated overnight. Next, the cells were treated with the indicated concentrations of F-NepA (0.2–3.2 μM) in growth media for the indicated times. Subsequently, the cells were gently washed twice with growth medium and incubated with 0.5 mg/mL MTT (Sigma-Aldrich) at 37 °C for 4 h. The formazan crystals formed by active mitochondria were dissolved in DMSO, and the absorbance in each well was measured at 570 nm and used to determine cell viability. The IC₅₀ values were calculated by a nonlinear regression analysis using TableCurve 2D v5.01 software (Systant Software Inc.). All reagents used in the MTT assay were purchased from Sigma-Aldrich.