Controlled cell adhesion assays Specific cell adhesion and spreading on coated surfaces were performed according to [28]. Briefly, to preserve cell-surface cadherins, twenty-four hours serum-starved GT1-7 cells were mechanical dissociated in trypsin-free conditions with PBS, 3.5 mM EDTA, 1.5% BSA on ice. Cells were then plated on the different adhesion substrates in serum-free conditions to prevent growth factors-mediated survival and at low cell density (8.104 cells/cm2) to prevent cell-cell contacts between neighboring cells. For experiments using inhibitors, cells were incubated 1 hour with the specific inhibitor prior to mechanically dissociation and throughout culture on the different substrates with inhibitors at the concentrations and times indicated. To induce Bad and Erk1/2 protein phosphorylation, confluent cells were treated with Phorbol 12-myristate 13-acetate (PMA) at 10 ng/ml for 1 hour. Calcium switch assays Confluent GT1-7 cell monolayers were deprived of serum for 24 hours, and the N-cadherin-mediated cell-cell contacts were disrupted by treatment with 4 mM EGTA in DMEM for 40 min at 37uC. The calcium-free medium was removed and N-cadherin contacts were allowed to re-establish by addition of DMEM, containing 1.8 mM Ca2+. The time of re-addition of Ca2+ was considered as 0 min. Following calcium restoration at 37uC, cells were harvested, lysed, protein extracts separated by SDS-PAGE and immunoblotted as described below. Alternatively, serum deprived GT1-7 cells were maintained in suspension in 1% agarose coated 60 mm culture dishes (10.104 cells/dish) to prevent cell attachment. Cells were either incubated in the presence of 1.8 mM Ca2+ (provided by the DMEM medium) or in the presence of DMEM plus 2.5 mM EDTA (0 mM free Ca2+) to prevent cadherin-dependent cell-cell adhesion. At various time points, cells were washed once with cold PBS, collected and processed for analysis of PARP cleavage by immunoblotting. Western blotting Cells were lysed in RIPA modified buffer (50 mM Tris-HCl, pH 7.4, 1% NP-40, 0.25% DOC, 150 mM NaCl, 20 mM sodium pyrophosphate, 50 mM sodium fluoride, 1 mM sodium orthova- nadate and protease inhibitor cocktail (Roche)). Lysates were vortexed 20 min at 4uC and then cleared by centrifugation at 10, 000 rpm for 15 min at 4uC. Equal amounts of proteins (20 mg determined by micro-BCA kit, Pierce) were loaded and separated by SDS-PAGE. After transfer at 4uC, nitrocellulose membranes were blocked with 5% nonfat milk in Tris-buffered saline, pH 8.0, containing 0.1% Tween 20 (TBST) prior to addition of the corresponding primary antibody and followed with IRDye- coupled secondary antibodies (Rockland) against rabbit or mouse immunoglobulins. For detection of phosphorylated proteins, antigens were detected with phospho-specific antibodies diluted in 5% BSA in TBST at 4uC. Protein bands were identified with Odyssey Imaging System (LI-COR Biosciences). Signals were quantified with LI-COR software. The membranes were reprobed with an anti-b-tubulin, anti-b-actin monoclonal or anti-pan Erk1/ 2 polyclonal antibodies for normalization. Immunocytochemistry and TUNEL assay Cytochrome c release was followed by immunostaining as described previously [37]. Briefly, at the end of the treatment time, cells were washed gently once with PBS followed by fixation in 4% formaldehyde for 15 min. The fixed cells were washed three times with PBS 5 min each, followed by permeabilization in 0.15% N-Cadherin Anti-Apoptotic Signaling Triton X-100 in PBS for 15 min. The cells were then blocked for 60 min in blocking buffer (2% BSA in PBS) followed by 4 hours incubation with a mouse monoclonal antibody against cytochrome c. The cells were washed three times for 10 min each in blocking buffer followed by 1 hour incubation with a rhodamine-labeled goat anti-mouse IgG (1/500, Jackson Immunology). The cells were washed three times for 10 min in PBS, counterstained with 0.5– 1 mg/ml DAPI, to examine nuclear morphology and quantify apoptosis. TUNEL assay was performed using the In Situ Cell Death Detection Kit, TMR Red (Roche Diagnostics) following the manufacturer’s protocol. Cells were viewed under a fluorescence microscope (Leica DM6000) and images were captured with a digital camera using Metamorph software. Different fields (at least 200 cells) were counted for each experiment. Statistical analysis Data were compared by ANOVA followed by protected t test for multiple comparisons. Paired sample means were compared using the t test. P values of 0.05 or less were considered statistically significant. Results N-cadherin engagement promotes survival of primary neurons In primary neuron culture studies, most culture protocols have been optimized to grow neurons on poly-ornithine (PO) or poly-L- lysine (PL) attachment factors. Associated culture media have been adapted by adding sera and/or B27 supplement. We observed that when these media additives were omitted from E18.5 rat hippocampus neurons cultured on PL, although cells initially bound to the plate, they rapidly died (Fig. S1). In contrast, replacing PL by recombinant Ncad-Fc (N-cad) as a substrate allowed maintaining healthy neurons in culture for at least 24 to 48 hours (Fig. S1). Although cells were seeded in similar conditions for each substratum, the observed reduction in healthy neurons on PL might be due to a deficit in the initial adhesion of these cells on PL. To assess this point, we counted the actual number of cells attached at 1 hour, 4 and 24 hours after seeding, and observed that there was no deficit of cell binding on PL compared to N-cad at early time points (Fig. S2A). This was confirmed by the fact that we were able to observe by time-lapse microscopy that cells initially attach then die (data not shown). To further characterize the effect of N-cadherin on survival of rat hippocampal neurons in the absence of B27 additive, cultures were fixed after 24 hours and stained for bIII-tubulin to identify neuronal cells. The total number of remaining bIII-tubulin positive cells was dramatically lower on the PL substrate (below 20%) in comparison to the number of cells remaining on the N- cad substrate (Fig. 1A, 2A), confirming that in these basal conditions, N-cad had a survival effect on primary hippocampal neurons. Similar observations were made for primary murine spinal cord cultures (Fig. 1B, 2B). In this case, the difference in the total number of bIII-tubulin positive cells between the PL and N- cad substrate was not as dramatic after 24 hours. However, DAPI staining revealed that the percentage of presumably apoptotic condensed nuclei was higher on PL (5462% of bIII-tubulin positive cells with condensed nuclei) than on N-cad substrates (2563% of bIII-tubulin positive cells with condensed nuclei). In addition, when we analyzed cell death by terminal deoxynucleo- tidyl transferase dUTP nick end labeling (TUNEL) we observed that the number of TUNEL positive cells in hippocampal cultures increased with time on PL whereas it remained very low on N-cad (Fig. S2, B). Altogether these observations show that primary PLoS ONE | www.plosone.org 3 March 2012 | Volume 7 | Issue 3 | e33206