Kinase experiment:

For Western blot analysis, 2N and 4N RPE1 cells are treated with DMSO, 50 ng/mL doxorubicin, or 1 μM DPBQ for 48 hours. Western blotting is performed with a SDS-PAGE Electrophoresis and semidry transfer. Samples are analyzed by chemiluminescence. β-actin is used as a protein loading control[1].

Cell experiment:

For Apoptosis assays, 2N and 4N RPE1 cells are treated with DMSO, 60 nM staurosporine, or 1 μM DPBQ for 48 hours. Apoptosis is evaluated by an Annexin V-PE/7-AAD apoptosis detection kit. For the flow cytometry analysis, the percent positive cells in the upper right and lower right quadrants are summed to give the total number of apoptotic cells. Three independent replicates are performed[1].

DPBQ is a p53 activator.

DPBQ elicits apoptosis and cell death and is selective for effects in 4N cells. DPBQ elicits expression and phosphorylation of p53 and this effect is specific to tetraploid cells. Knockdown of TP53 restores proliferation of tetraploid cells in the presence of DPBQ[1].

[1]. Choudhary A, et al. Identification of Selective Lead Compounds for Treatment of High-Ploidy Breast Cancer. Mol Cancer Ther. 2016 Jan;15(1):48-59.