2.2. Cell Viability Assay

Cell viability assay was performed using CellTiter-Glo Luminescent Cell Viability Assay (Promega, Madison, WI, USA) following manufacturer’s instructions. In brief, for each cell line, a cell suspension containing 5000 to 7500 viable cells was seeded in 96-well plates 24 h before treatment with either the small kinase MEK inhibitor Selumetinib (AZD6244, Selleckchem, Houston, TX, USA), the Cdk 4/6 inhibitor Palbociclib (Selleckchem, Houston, TX, USA) or the thiazole antibiotic known to modulate FoxM1 expression and transcriptional activity Siomycin (Cayman Chemical, Ann Arbor, MI, USA). Number of plated cells was selected based on each line’s proliferation rate. Compounds were dissolved in dimethyl sulfoxide (DMSO) (Selumetinib and Siomycin) or phosphate-buffered saline (PBS) (Palbociclib), and cells were treated in a 2-fold serial dilution curve ranging from 0.15 μM to 150 μM for Selumetinib, from 1.12 μM to 10 μM for Palbociclib and from 0.25 μM to 1.25 μM for Siomycin. Matched PBS and DMSO control data were collected for each dilution point. Independent biological replicates (n = 4) were collected for each cell line. After 72 h of incubation with the compounds, plates were brought to room temperature for 30 min. Media were replaced with a 1:1 solution of CellTiter-Glo and fresh media, and cells were lysed on an orbital shaker at room temperature for 5 min. Luminescence signal was measured using a Beckman Coulter DTX 880 microplate reader (Beckman Coulter, Brea, CA, USA) [19].