4.3.1. Western Blot for Phospho-γH2AX Histone

MCF-7 breast cancer cells were seeded at a density of 1.0 × 10⁵ cells/well in a 12-well plate and allowed to settle for 48 h before exposure to PK083, PK9320, or DMSO. At the end of the treatments, the cells were washed with phosphate-buffered saline and lysed using 1x SDS-loading buffer containing β-mercaptoethanol (5x buffer: 0.25% w/v bromophenol blue, 0.5 M DTT (dithiothreitol), 50% v/v glycerol, 10% v/v SDS (sodium dodecyl sulphate) and 0.25M Tris-HCl, pH 6.8). The lysates were sonicated and clarified by centrifugation at 13,000 r.p.m. before being loaded into 12% SDS-polyacrylamide gels. Proteins were separated by SDS-PAGE at 200 V for 35 min. Western blot analysis of phospho-γH2AX histone and the loading control α-tubulin was accomplished through transfer of protein onto PVDF membranes using the Trans-Blot Turbo System (Bio-Rad, Hercules, CA, USA). Mouse monoclonal anti-α-tubulin (DM1A) was purchased from Abcam (Cambridge, UK) and mouse anti-phospho-histone H2AX (Ser139) (JBW301) was purchased from Millipore (Darmstadt, Germany). Secondary antibodies were HRP-conjugated, polyclonal goat-derived antibodies against mouse and rabbit (Dako, Agilent Technologies, Santa Clara, CA, USA). After blotting and immunoreactions, protein bands were detected with a chemiluminescent Western blotting substrate (SuperSignal West Femto, ThermoFisher) and the ImageQuant LAS 4000 system (GE Healthcare, Chicago, IL, USA). Protein bands were quantified by densitometry using ImageJ 1.50C and phospho-histone H2AX bands were normalized to the α-tubulin control band. DNA damage was reported as the percent of normalized phospho-histone H2AX. DNA damage assays were repeated in triplicate and the half maximal effective concentration (EC₅₀) value for PK9323 compound compared with that of cisplatin as a reference DNA lesion-inducing drug using the non-linear regression module of Prism 6.0 (GraphPad, San Diego, CA, USA). In light of the results obtained from the dose–response assays with PK9323, the following experiments were carried out for 20 h in the presence of either 2.2 (PK9323 EC₅₀) or 10 μM (maximum concentration tested) of either of the three compounds; both working concentrations were obtained by dilution of either stock solution with a constant volume of DMSO. Negative controls were prepared by exposing cells to the same volume of DMSO vehicle only for 20 h.