Events leading to apoptotic death in BT474 and SKBR3 breast cancer cells treated with human recombinant β galactoside binding protein (Hu-r-βGBP). (a-b)Rate of cell proliferation and dose response to Hu-r-βGBP. Dotted lines in growth curves define the time of occurrence of apoptotic events. Values are means of triplicate cultures ± standard error of the mean (SEM). (c-d)Sequential expression of apoptotic events after treatment with 30 nM Hu-r-βGBP. Top to bottom: loss of mitochondrial membrane potential assessed by tetramethylrhodamine ester (TMRE); phosphatidylserine orientation at the plasma membrane assessed by annexin V staining; caspase 3 activity and DNA fragmentation (terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labelling (TUNEL)). Apoptotic cells are in circled areas. Values are percentages of cells in apoptosis. Left panels: controls, right panels: cells treated with 30 nM Hu-r-βGBP. (e, h)Inhibition of class IA phosphoinositide 3-kinase (PI3K) activity by 30 nM Hu-r-βGBP measured as phosphatidylinositol (3,4,5)-trisphosphate (PIP3) generated from immunoprecipitated PI3K in a kinase assay at 30°C, and assessed in a competitive ELISA. Values are means from triplicate readings ± SEM. (f, i) akt mRNA and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA levels. Left panels: controls, right panels: cells treated with 30 nM Hu-r-βGBP. (g, j)Phosphorylated (Ser473) Akt and total Akt protein. Left panels: controls, right panels: cells treated with 30 nM Hu-r-βGBP. (a-j) Hu-r-βGBP added at 3 h after seeding. The data are representative of results obtained in three separate experiments.