N-Cadherin Anti-Apoptotic Signaling Figure 5. N-cadherin engagement protects neuronal cells from apoptosis. GT1-7 cells were cultured as indicated in Figure S2 for 24 (A, B) or 4 hours (C) and processed for nuclear condensation (A), cytochrome c release (B) as well TUNEL libeling (C). PARP cleavage was analyzed by western blotting of equivalent protein content from cell lysates (D). Results in A, B and C are expressed as the mean 6 SD of four independent experiments. Western blotting in D is representative of three independent experiments. Asterisks (*) or (***) indicate a significant difference (P,0.05) or (P,0.01) respectively as compared with PL. doi:10.1371/journal.pone.0033206.g005 cadherin engagement. Several studies have reported that, during ano¨ıkis, Bcl-2 family members such as the anti-apoptotic Bcl-2 and Bcl-xL proteins or the pro-apoptotic proteins Bad or Bim are regulated by activated MAP Kinases [10]. Therefore, we measured the Bcl-2 protein levels by immunoblotting at several time points. Bcl-2 protein levels remained constant after N- cadherin engagement and at levels equivalent to those of cells seeded on PL (Fig. 9A). Consistent results were obtained with the calcium switch assay (Fig. 9B), suggesting that cell survival induced by N-cadherin engagement was not mediated by an increase of the anti-apoptotic Bcl-2 protein levels. It has also been demonstrated that the early phosphorylation of the pro-apoptotic protein Bad on Ser-112 by MAP kinases leads to its inactivation [41] resulting in cell survival. However, we did not detect any increase of Bad phosphorylation in cells seeded on N- cad at the earlier time point (4 hours) as compared to cells seeded on PL (Fig. 9C). Moreover, calcium restoration in confluent GT1- 7 cells did not induce Bad phosphorylation at any time point tested (Fig. 9D). These results indicate that Bcl-2 and Bad proteins take not part in the survival pathway induced by N-cadherin mobilization. N-cadherin engagement down-regulates the pro- apoptotic protein Bim-EL The Bim protein is another critical regulator of apoptosis downstream of MAP kinase signaling. We determined whether N- cadherin engagement affects Bim protein levels. In confluent GT1- 7 cells, Bim was expressed at low levels and Bim-EL was the only form of Bim detected. However in cells deprived of cell-matrix and cell-cell contacts (PL substrate) Bim-EL was up-regulated, while it remained low in cells seeded on N-cad. At 24 hours Bim-EL protein level was 3.5 fold higher in cells seeded on PL than on cells seeded on N-cad (Fig. 10A), suggesting that N-cadherin engage- ment might favor cell survival by down-regulating Bim-EL. To ascertain whether Erk1/2 pathway acts downstream of N- cadherin to regulate the level of Bim-EL protein, N-cad seeded cells were treated with 20 mM U0126. This treatment induced a 3 fold increase in Bim-EL levels thereby reaching similar levels to those in cells seeded on PL (Fig. 10A). The increase of this pro- apoptotic protein was consistent with the raise of the apoptotic index in cells seeded on N-cad and treated with U0126 (Fig. 8). Altogether these findings indicate that MAP kinase activation following N-cadherin engagement could sustain GT1-7 cell survival by maintaining Bim-EL protein levels low. Interestingly, we observed that adhesion of GT1-7 cells to the pro-survival FN substrate down-regulates Bim-EL in a similar Erk1/2-dependent manner than N-cadherin (Fig. S8). Luciano et al. showed that phosphorylation of Bim-EL by Erk1/2 on Ser-69 triggers its degradation by the proteasome pathway and promotes cell survival [33]. We thus searched for changes in the status of Bim- EL phosphorylation. As a positive control of Bim-EL phosphor- ylation, cells were treated with PMA. Addition of PMA induced a PLoS ONE | www.plosone.org 8 March 2012 | Volume 7 | Issue 3 | e33206