Cell Viability Assay with MTT

Cell viability was tested by MTT assay (Sigma, St. Louis, MO, USA). Briefly, cells were seeded in 96-well plates (4 × 10³ cells/well) and were treated the following day for 48 h with erlotinib, nelfinavir or nitroxoline as single agents, or with combinations of the drugs at various concentrations as indicated. Then, the MTT solution was added to each well and incubated at 37 °C for at least 3 h, until a purple precipitate was visible. In order to dissolve formazan crystals, the culture medium was replaced with dimethyl sulfoxide (DMSO, Euroclone). Absorbance of each well was quantified at 540 and 690 nm, using a Synergy H1 microplate reader (BioTek Instruments Inc., Winooski, VT, USA).