Figure 5

Calcium entry into mitochondria is driven by a BclxL/VDAC1 interaction, which promotes cell motility. (a) Bclx-KO MEFs reconstituted with BclxL-MT (MEF^BclxL-MT) or BclxL-ER (MEF^BclxL-ER) were stimulated with cl-CD95L (100 ng/ml) for the indicated amount of time. Cells were lysed and BclxL was immunoprecipitated. The immunoprecipitated complex was resolved by SDS-PAGE and immunoblotted with anti-BclxL and anti-VDAC1 antibodies. MEF^BclxL−/− were used as a negative control. (b) The experiment in (a) was performed with BT549^ShBclx reconstituted with BclxL-MT or BclxL-ER. (c) BclxL-deficient MEFs reconstituted with BclxL-WT were loaded with the mitochondrial Ca²⁺ probe (Rhod2) and cell-permeable Mitotracker Green. Representative graphs (10 cells) depicting the relative mitochondrial calcium concentration in control cells (left panel) and L14-15- (middle panel) and N-Ter (right panel)-treated cells (1 μM, 1 h) are shown. (d) BclxL-KO MEFs reconstituted with BclxL-WT (MEF^BclxL-WT) or BclxL-MT (MEF^BclxL-MT) were pre-incubated with the indicated cell-penetrating peptides (1 μM) for 1 h and then stimulated with or without cl-CD95L (100 ng/ml). Cell migration was evaluated by the Boyden chamber assay. Migrating cells were fixed and stained with Giemsa. For each experiment, five images of random fields were acquired. To quantify cell migration, migrating cells were lysed and absorbance was measured at a wavelength of 560 nm. Values represent the mean±standard deviation of three independently performed experiments (**P<0.01, *P<0.05; two-way Mann-Whitney)