Effect of ALE on the clonogenic growth of DBF4-downregulated MCF-7 cells. (a) Endogenous DBF4 protein was downregulated by siRNA. MCF-7 cells were transfected with only oligofectamine (lane 1, mock), 40 nM control siRNA Luciferase GL2 Duplex (lane 2), and 40 nM of siGENOME duplex pool directed against DBF4 (lane 3). The total protein extracts were subjected to SDS-PAGE and DBF4 protein levels were quantified by western blotting (WB) and actin was measured as loading control. Five hours after siRNA transfection, MCF-7 cells were subjected to ALE treatment at a concentration of 10-6 M for 48 h. (b) Following stimulation, 1,000 cells were plated for the clonogenic assay. After 10 days, the colonies were stained with 10% crystal violet and scored using ImageQuant TL computer software. The experiments were performed in triplicates and the error bars represent standard error of the mean. Black bars represent untreated cells, while stripped bars correspond to DBF4-downregulated cells. Ctrl, control.