Differential Regulation of Growth-Promoting Signalling Pathways by E-Cadherin
Figure 2
Calcium-induced increase in proliferation in low-density NHU cultures occurs via activation of the PI3-K/AKT pathway.
(A) NHU cells were seeded in medium containing low [Ca2+] (0.09 mM) and left to attach overnight. Calcium concentration was then increased to 2 mM and protein lysates were prepared at the indicated time-points for immunoblotting. Expression of phospho-AKT (green) and total AKT (red) was determined using rabbit and mouse antibodies, respectively, followed by fluorochrome-conjugated secondary antisera as in Figure 1C. Immunolabelling was visualised and densitometry to determine fold induction of p-AKT expression with respect to total AKT was performed as in Figure 1C. (B) NHU cells were cultured in medium containing 0.09 mM (Low Ca2+) or 2.0 mM (Phys Ca2+) [Ca2+]. Expression of phospho-AKT (p-AKT) was determined by immunofluorescence microscopy using anti-p-AKT rabbit antibody followed by goat anti-rabbit antibody conjugated with Alexa Fluor 488 (green). Cell nuclei were visualised using Hoechst 33258 (blue). (C) NHU cells were seeded into 96-well plates and cultured for a period of 7 days in medium containing either low (-Ca) or physiological (+Ca) calcium levels, in the presence or absence of 5 µM of the PI3-K inhibitor LY294002. Proliferation was determined on the basis of cell biomass using the MTT assay. Data points represent mean absorbance values for 6 replicate wells (±S.E.M.). Results for all data points are also presented in the form of bar graphs (lower panel) for the purpose of statistical analysis. ns, non-significant; **, P<0.01; ***, P<0.001.