4.5. Cell-Based Assays

For each cell line and each inhibitor, we performed proliferation, viability, senescence, colony formation, and cell cycle analysis, as described below.

To evaluate cell proliferation, the TACS MTT Cell Proliferation Assays (Trevigen, Gaithersburg, MD, USA) were used. The RT cell lines (MON, BT-12, and BT-16) were plated at 2 × 10³ cells on each well of a 96-well plate. The MB cell lines (DAOY and D283) were plated at 5 × 10⁴ and 5 × 10⁵ cells per well, respectively. The absorbance was measured after 24, 48, 72, and 96 h at concentrations ranging from 0.001 to 10 µM of the inhibitor. All assays were performed in triplicates.

Cell viability was assessed using the Presto Blue™ Cell Viability reagent (Thermo Fisher Scientific, Carlsbad, CA, USA) following the manufacturer’s instructions. Cells were plated in 96-well plates at the same densities described above for the proliferation assay. The fluorescence was measured after 24, 48, 72, and 96 h at concentrations ranging from 0.001 to 10 µM of the inhibitor. All assays were performed in triplicate.

All cell lines were seeded at 200 cells per well in six-well plates and incubated overnight. After 24 h, the treatment medium was added and changed once a week. Cells were treated with 0.01, 0.05, 0.1, 0.2, 0.5, 1, 5, and 10 µM of PLK4i and 0.1% DMSO as control. After 14 days, the cells were washed twice with 1x PBS, fixed with formalin, and stained with Cresyl violet (ACROS Organics, Pittsburgh, PA, USA). Colonies were counted using the ImageJ software (www.imagej.nih.gov, accessed on April 21, 2017).

Senescence was evaluated using the Beta-galactosidase assay (Senescence Cells Histochemical Staining Kit CS0030 (Sigma, St Louis, MO, USA) and clonogenic recovery assay as previously described.

Cell cycle analysis was performed by flow cytometry of cells stained with Propidium Iodide (PI) (Thermo Fisher Scientific, Carlsbad, CA, USA) according to manufacturer’s instructions. Cells treated with the top PLK4i candidates and 0.1% DMSO (control) were fixed in 80% ethanol overnight, stained with PI, and then subjected to flow cytometric analysis using a BD Fortessa instrument (BD Biosciences, San Jose, CA, USA). Data was analyzed using Modfit LT from Verity Software House (Topsham, ME, USA). All experiments were performed in triplicate.