N-Cadherin Anti-Apoptotic Signaling Figure 9. N-cadherin engagement does not affect Bcl-2 protein level and Bad phosphorylation. Serum-starved GT1-7 cells were seeded at low density on PL or N-cad and grown for the indicated time (A) or for 4 hours (C) in serum-free medium. In B-D, cells were subjected to a calcium switch protocol. As a positive control, cells were treated with 10 ng/ml of PMA or DMSO for 1 hour. Equivalent amount of proteins were immunobloted with antibodies to Bcl-2 and phospho-Bad. Relative densities of Bcl-2 and phospho-Bad were normalized with either a-tubulin, or b- actin. Results are the mean 6 SD of three independent experiments. doi:10.1371/journal.pone.0033206.g009 We then analyzed the effect of Bim-EL S69G overexpression on the survival of cells seeded on N-cadherin by determining the percentage of transfected cells with a condensed nucleus 6 hours and 24 hours after plating (Fig. 11B). The expression of GFP alone did not lead to an increase in the occurrence of dying cells compared to un-transfected cells plated on N-cadherin (compare with Figure 5). In contrast, the overexpression of Bim-EL S69G induced a significant increase in the number of apoptotic cells both at 6 and 24 hours. The overexpression of the wild type form of Bim-EL expressed at similar levels (Fig. 11A) had no significant effect on GT1-7 cells plated on N-cadherin at 6 hours, although it impaired the survival at 24 hours (Fig. 11B), suggesting that the accumulation with time of wild time Bim-EL may also lead to apoptosis due to the overwhelming of the Bim-EL phosphorylation and degradation pathway. Altogether, these data indicate that the accumulation of Bim-EL and in particular of its Erk- phosphor- ylation defective mutant (S69G) prevents N-cadherin-induced cell survival, supporting the hypothesis that N-cadherin triggers neuronal cell survival through a Bim-EL down-regulation. Discussion In this study, we accumulate convergent arguments to support the hypothesis that N-cadherin engagement provides neuronal cells with a pro-survival signal. We cannot formally exclude the contribution of other factors or adhesion molecules to this process. However, the anti-apoptotic effect of N-cadherin engagement was observed in tightly controlled conditions, that is in isolated cells, in the absence of growth factors, and in the absence of detectable integrin activation in focal adhesion complexes. Furthermore the survival effect of the N-cad substrate was alleviated by the expression of a dominant negative form of N-cadherin. These results strongly support the hypothesis that N-cadherin initiates an anti-apoptotic pathway in neurons and neuronal cells that is PLoS ONE | www.plosone.org 11 March 2012 | Volume 7 | Issue 3 | e33206