N-Cadherin Anti-Apoptotic Signaling Figure 3. Inhibition of calcium-dependent cell-cell adhesion induces apoptosis of GT1-7 cells in suspension. Serum-starved GT1-7 cells were cultured in suspension in agarose-coated dishes in the presence of calcium (1.8 mM) or without calcium (2.5 mM EDTA). (A) Phase-contrast photomicrographs were taken at time 0 and 24 hr of incubation. (B) Cells lysates were then analyzed by western blotting for PARP cleavage. Western blot representative of two independent experiments. doi:10.1371/journal.pone.0033206.g003 accumulated at focal adhesions in GT1-7 cells seeded on FN (Fig. S4 and data not shown). To confirm that the survival effect was the result of a specific engagement of N-cadherin, we tested the ability of a dominant negative mutant form of N-cadherin [32] to perturb cell survival triggered by N-cadherin. This DN-Ncad mutant was expressed in GT1-7 cells that were then seeded on N-cad substrate. While most of the control GT1-7 cells transfected with a GFP-encoding plasmid displayed healthy nuclei morphology after 24 hours on the N-cad substrate, more than 50% of the DsRed positive cells displayed condensed nuclei (Fig. S5), indicating that the expression of DN-Ncad counteracts the protective effect of the N-cad substrate. These results suggest that N-cadherin engagement protects cells against apoptosis in a specific manner. To gain insight on the specific cell death pathway activated, cytochrome c release from mitochondria to the cytosol was evaluated by immunostaining (Fig. 4). After 24 hours, a vast majority of cells (80%) seeded on PL showed a cytochrome c distribution characteristic of cytosolic localization and very often a total loss of cytochrome c, as reported in [38], (Fig. 5B). In contrast, most of the cells seeded on N-cad showed a punctuated or tubular perinuclear pattern characteristic of the mitochondria network and only 22% of the cells had released their cytochrome c out of mitochondria. Similar results were obtained with cells seeded on FN (positive control of cell survival), with less than 20% of cells showing diffuse cytochrome c staining (Fig. 5B). Time course studies showed that nuclear condensation and cytochrome c release were already high 6 hours after plating cells on PL whereas the percentage of apoptotic figures remained low and constant for cells on N-cad (Fig. S6). To confirm that these cytological changes were part of an ongoing apoptotic process that was repressed by N-cadherin PLoS ONE | www.plosone.org 6 March 2012 | Volume 7 | Issue 3 | e33206