Cell proliferation and growth assays

Cells were seeded in 96-well plates at a density of either 7000 cells (H3122, 24-H3122 and CR-H3122) and 4000 cells (A549) and allowed to adhere for 24 hours. Cells were treated with individual drugs alone or drug combinations for 72 hours before assay. Cytotoxicity and proliferation rate was evaluated using the sulforhodamine B (SRB) assay, as described by Skehan et al. (1990). Briefly, cells were fixed with 50 µL 10% trichloroacetic acid (TCA) for 30 minutes at 4 °C. Protein was stained with 50 µL of SRB and then the wells washed with 1% acetic acid. The plate was then dried and SRB solubilised in 100 µL of 100 μM TRIS buffer (Sigma-Aldrich, US). Absorbance was read at 490 nm with a Spectromax plate reader, deducting the background of 630 nm; cell growth inhibition was evaluated as the ratio of the absorbance of the treated cells with the DMSO-treated control. All cytotoxicity assays were carried out in three independent experiments measured in technical triplicate. Cell proliferation time points were measured in hextuplicate.