E-Cadherin Growth Regulation 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 mM 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 (6S.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. doi:10.1371/journal.pone.0013621.g002 formation of cell-cell contacts and its potential involvement in the regulation of such signalling pathways, we performed functional inactivation of E-cadherin in NHU cells by transduction with a retrovirus expressing the dominant-negative E-cadherin construct H-2Kd-E-cad [22,23]. The construct encodes a chimeric protein comprising the extracellular domain of H-2Kd class I and the transmembrane and intracellular domains of E-cadherin. Recom- binant retrovirus expressing the E-cadherin mutant was used to infect NHU cells alongside a control virus, giving rise to isogenic sub-lines NHU-ECmut and NHU-Con, respectively. As our optimised retrovirus method for stable NHU cell transduction is .90% efficient [24], NHU sub-lines were derived as non-clonal populations, thus negating integration artefacts. By flow cytometry detection, the expression of surface H-2Kd-E-cad on NHU- ECmut cells was very low (Figure 3A, upper panels), but immunofluorescence microscopy showed substantial intracellular expression, with the H-2Kd-E-cad protein localising mainly to perinuclear areas (Figure 3A, lower panels). In low calcium medium, NHU cells showed moderate, diffuse cytoplasmic E-cadherin expression, which became intense and localised to intercellular borders within hours of switching to physiological calcium (Figure 3B). Associated with the calcium- mediated stabilisation of E-cadherin, the major cytoplasmic- domain partners of E-cadherin were also relocated to different intracellular compartments. Thus, a-catenin showed a change from diffuse cytoplasmic to intense plasma membrane and cytoskeletal filament localisation, whereas b-catenin relocalised from the nucleus to cell-cell contact points (Figure 3B). Expression of the dominant-negative E-cadherin construct H-2Kd-E-cad caused a dramatic change in the pattern of endogenous E- cadherin localisation. In the retrovirus-transduced NHU-ECmut cells maintained in 2 mM calcium, E-cadherin was detected in PLoS ONE | www.plosone.org 4 October 2010 | Volume 5 | Issue 10 | e13621