Figure 2

Effects of cholera toxin (100 ng/ml) and RasV12 on MCF10A breast ductal cells and response to treatment with human recombinant β galactoside binding protein (Hu-r-βGBP). (a-c) Cell proliferation and dose response to Hu-r-βGBP. Values in growth curves are means from triplicate cultures ± standard error of the mean (SEM). (d-f) Apoptotic response to treatment with 30 nM Hu-r-βGBP. Apoptosis assessed by annexin V staining at day 3 after seeding. Apoptotic cells are in circled areas. Values are percentages of cells in apoptosis. Left panels: controls, right panels: cells treated with Hu-r-βGBP. (g-i) Inhibition of Class IA phosphoinositide 3-kinase (PI3K) by 30 nM Hu-r-βGBP assessed as described in Figure 1. Values are means from triplicate readings ± SEM. (j-l) akt mRNA and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA levels. Left panels: controls, right panels: cells treated with 30 nM Hu-r-βGBP. (m-o) Phosphorylated (Ser473) Akt and total Akt protein. Left panels: controls, right panels: cells treated with 30 nM Hu-r-βGBP. (a-o) Hu-r-βGBP added at 3 h after seeding. The data are representative of three separate experiments. Cholera toxin was used to enhance mitogenic signalling via extracellular signal-regulated kinase (ERK) upregulation [35]. Constitutively expressed RasV12 was an endogenous source of enhanced mitogenic signalling [36].