N-Cadherin Anti-Apoptotic Signaling Figure 8. Mek 1/2 inhibitor induces apoptosis on cells seeded on N-cadherin. Serum-starved GT1-7 cells were pretreated with 20 mM U0126 or vehicle for 1 hour, then seeded at medium density on N-cad with or without 20 mM U0126 for 6 hours (A). Apoptosis was evaluated by determination of nuclei condensation following DAPI staining and cytochrome c release following immunostaining. Arrows point toward cells showing condensed nuclei and absence of cytochrome c staining while asterisks indicate a cell with healthy nuclei and cytochrome c staining. Scale bar: 20 mm. (B, C). The percentage of cells with condensed nuclei and diffuse/absent cytochrome c labeling was quantified at 6 and 24 hours in treated and non-treated cells seeded on PL or N-cad. Results are expressed as the mean 6 SD of four independent experiments. Asterisks indicate a significant difference (P,0.05) as compared with PL. doi:10.1371/journal.pone.0033206.g008 Overexpression of a S69G phosphorylation defective form of Bim-EL prevents N-cadherin cell survival In order to test whether the phosphorylation and down- regulation of Bim-EL was involved in mediating N-cadherin engagement-induced cell survival, we looked at the effect of overexpression of an Erk-dependent phosphorylation site defective mutant of Bim-EL (Bim-EL S69G) [33] on the survival of GT1 cells seeded on N-cadherin (Fig. 11). To prevent massive accumulation of the pro-apoptotic protein in cells before their settlement on N- cadherin, Bim-EL expression vector were lipofected 2 hours before plating on the N-cadherin substrate. To identify transfected cells, the Bim-EL vector was lipofected together with a GFP expressing vector. We first assessed the expression of Bim-EL S69G protein in confluent GT1-7 cells 8 hours and 24 hours post-transfection (Fig. 11A). While low levels of endogenous Bim-EL were detected in GFP alone transfected cells, a 2.5 to 3 fold increase in Bim expression was observed in Bim-EL S69G transfected cell cultures, for a percentage of transfected cells around 10% of the cell culture population, indicating that the Bim-EL mutated protein indeed highly accumulates in individual transfected cells. PLoS ONE | www.plosone.org 10 March 2012 | Volume 7 | Issue 3 | e33206