Cell Viability Assays

For the pharmacological studies, Lapatinib (cat. no. HY-50898) was purchased from MedChem Express and Deferiprone (3-hydroxy-1,2-dimethyl-4(1H)-pyridone) from Sigma-Aldrich.

HCC-1954, SKBR3, BT474 and MCF-10A cells were seeded at a density of 5 × 10³ cells per well on 96-well plates. After 24 h incubation (37°C, 5% CO₂), the cell medium was removed, and the cells were treated with various concentrations of Lapatinib (10 nM-30 μM) and deferiprone (10 nM-30 μM), either alone or in combination for 72 h. The medium was then removed and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution (0.3 mg/mL in PBS) was added to cells for 3 h, after which the MTT solution was removed and the formazan crystals were dissolved in 100 μL DMSO. The optical density was measured at 570 nm and at a reference wavelength of 650 nm using an absorbance microplate reader (SpectraMax 190, Molecular Devices, Sunnyvale, CA, USA). The 50% cytostatic concentration (IC₅₀) was calculated based on a four-parameter logistic equation using GraphPad Prism 5 (GraphPad Software, Inc.).