E-Cadherin Growth Regulation Figure 1. Physiological calcium causes a transient increase in proliferation in low density NHU cultures which is independent of EGFR/ERK signaling. (A) NHU cells were seeded in 6-well plates at 26104/well in medium containing 0.09 mM (Low Ca2+) or 2.0 mM (Phys Ca2+) extracellular [Ca2+]. Cell growth was determined by cell counting for a total period of 9 days. Each data point represents the mean (6S.E.M.) of 3 replicates (n = 3) and results are representative of at least three independent experiments. ns, non-significant; *, P,0.05; **, P,0.01. (B) NHU cells were cultured as above and expression of EGFR, phospho-ERK (pERK) and total ERK was assessed by immunofluorescence microscopy using rabbit (EGFR and p-ERK) and mouse (total ERK) antibodies, followed by goat antisera conjugated with Alexa Fluor 488 (green) or 594 (red). Cell nuclei were visualised using Hoechst 33258 (blue). (C) NHU cells were seeded in standard culture medium (0.09 mM calcium) and left to attach overnight. Calcium concentration was then increased to 2 mM and protein lysates were prepared at the indicated time-points for gel-electrophoresis and immunoblotting. Expression of EGFR, phospho-ERK (pERK), total ERK and b-actin was determined using mouse antibodies (total ERK/b-actin) and rabbit antisera (EGFR/pERK), followed by goat anti-mouse antibody conjugated with Alexa Fluor 680 (red) or goat anti-rabbit antibody conjugated with IRDye 800 (green). Densitometry was performed to determine fold induction of EGFR and pERK expression following normalisation against b- actin and total ERK, respectively. Bar graphs represent results relative to the expression level of the initial time point (0 hours). doi:10.1371/journal.pone.0013621.g001 PLoS ONE | www.plosone.org 3 October 2010 | Volume 5 | Issue 10 | e13621