Cell viability through MTT assay

In order to evaluate gefitinib sensitivity, cells were seeded at a density of 1 × 10⁴ cells/well into a 96-well plates and after 24 h were treated with various gefitinib concentrations (0, 0.01, 0.1, 1, 10 μM).

After 72 h at 37°C, the medium was replaced with a complete medium without phenol red and 10 μl of 5 mg/mL MTT [3-(4,5-dimethylthiazol-2)-2,5-diphenyltetrazolium bromide] solution (Sigma, St. Louis, MO, USA) were added to each well. After a further 4 h incubation time, absorbance upon formed formazan crystals solubilisation with 10% Triton-X-100 in acidic isopropanol (0.1 N HCl) was measured at 570 nm using a micro plate reader. Viabilities were expressed as a percentage of the untreated controls. The 50% growth inhibition (IC₅₀) was determined from dose-response curve. Each experiment was performed in three replicate wells for each drug concentration and results are presented as the median of at least three independent experiments.

In order to assay the effect of NEU3 on cell viability in the presence of 27 nM or 1 μM gefitinib, cells were seeded in 96-well plates at a density of 1 × 10⁴ cells/well and after 24 h were transiently transfected. After 4 h from the addition of the jetPEI/DNA mixture to the cells, the medium was changed and the cells were treated with 27 nM or 1 μM gefitinib. After an incubation at 37°C for 36 h post transient transfection, the medium was replaced with complete medium without phenol red and 10 μl of 5 mg/ml MTT solution were added to each well. After a further 4 h incubation time, absorbance upon solubilisation was measured at 570 nm using a micro plate reader. Viabilities were expressed as a percentage of the mock.