E-Cadherin Growth Regulation Figure 4. Loss of E-cadherin function enhances NHU cell proliferation and activates the EGFR/ERK and b-catenin/TCF signalling pathways. (A) NHU-Con and NHU-ECmut cells were seeded in 96-well plates and cultured as above. [3H]-thymidine (TdR) precursor was added 24 hours later and 16 hours post-pulsing, cells were harvested and TdR uptake measured by scintillation spectrometry. Data represent mean of cpm counts (6S.E.M.) for 12 replicate wells. ns, non-significant; *, P,0.05; **, P,0.01. Of note, NHU-Con cells showed higher proliferation levels in low calcium compared to physiologic calcium conditions, hence there was no apparent calcium-mediated enhancement in growth described above. This is because [3H]-thymidine incorporation studies assessed proliferation in NHU cultures that were at .50% confluence, thus missing the initial phase where cells exhibit calcium- mediated increased growth at low-density. (B) NHU-Con and NHU-ECmut cells were cultured in medium containing 0.09 mM (low Ca) or 2.0 mM (phys Ca) [Ca2+] and proliferation assessed by cell counting as in Figure 1A. Each data point represents the mean (6S.E.M.) of 3 replicates and results are representative of at least two independent experiments. Results for days 3, 5 and 7 are also presented in the form of bar graphs (lower panel) for the purpose of statistical analysis and clarity. ns, non-significant; *, P,0.05; **, P,0.01; ***, P,0.001. (C) NHU-Con and NHU-ECmut cells were cultured in medium containing low (-Ca2+) and physiological (+Ca2+) calcium levels for 24 hours and protein lysates were prepared. Expression of phospho-ERK (p-ERK) and -AKT (p-AKT) as well as total ERK and AKT was determined using primary and secondary antibodies described in Figures 1C and 2A. Densitometry was performed to determine fold induction of p-ERK and p-AKT expression following normalisation against total ERK and total AKT, respectively. Bar graphs represent results relative to the expression level for NHU-Con in low calcium (-Ca2+). (D) NHU-Con and NHU-ECmut cells were transfected with the TCF/LEF firefly luciferase reporter TOPflash or the FOPflash control plasmid, alongside the Renilla luciferase expression vector pRL-tk. Four hours post-transfection, culture supernatants were adjusted for the desired level of calcium concentration, i.e. 0.09 mM (Low Ca2+) or 2.0 mM (Phys Ca2+). Cell lysates were prepared and reporter activity assessed in a 96-well format using the Dual-Luciferase Reporter Assay on a microplate reader for pair-wise detection of luminescence activity for each reporter. Firefly luciferase values were normalised against those of Renilla luciferase and were then expressed as fold activity with respect to that obtained for FOPflash in NHU-Con cells cultured in low or physiological calcium. Bars represent mean fold Firefly luciferase activity (6S.E.M.) for 6 replicate samples. doi:10.1371/journal.pone.0013621.g004 PLoS ONE | www.plosone.org 7 October 2010 | Volume 5 | Issue 10 | e13621