Figure 4

Localization of BclxL in the outer mitochondrial membrane is pivotal to induce CD95-mediated cell migration. (a) BT549 cells infected with a shRNA targeting the Bclx 3′ untranslated region (ShBclx#54) were reconstituted with BclxL-WT, BclxL-ER, or BclxL-MT devoid of the 3′ untranslated region. Cells were lysed and the expression levels of the indicated proteins were evaluated by immunoblotting. (b) Cells in (a) were loaded with the Ca²⁺ probe Fluo2 and stimulated with cl-CD95L (100 ng/ml). [Ca²⁺]_i was monitored via the ratio F/F₀ (relative Ca²⁺_[CYT]). Data represent the mean±s.e. of the mean of F/F₀ in GFP-positive cells (n>50 cells, at least three independent experiments). (c) Migration of cells depicted in (a) was analyzed by the Boyden chamber assay for 24 h in the presence (100 ng/ml) or absence of cl-CD95L. Migrating cells were stained with Giemsa and lysed. Absorbance was measured at a wavelength of 560 nm. Values represent the mean±standard deviation of three independently performed experiments (*P<0.05; two-way Mann-Whitney). (d) Bclx-deficient BT549 cells (BT549^ShBclx) reconstituted with BclxL-MT were loaded with the Ca²⁺ probe Fluo2 and pre-incubated for 1 h in the presence or absence of a non-toxic dose of ABT-199 (1 μM). Cells were then stimulated with 100 ng/ml cl-CD95L (black arrow), and [Ca²⁺]_i was monitored via the ratio F/F₀ (relative Ca²⁺_[CYT]). Data represent the mean±standard error of the mean of F/F₀ in GFP-positive cells (n>50 cells, at least three independent experiments). (e) BT549^ShBclx-BclxL-MT were pre-incubated for 1 h with or without ABT-199 (1 μM) and then treated or untreated with cl-CD95L (100 ng/ml) for 24 h. Cell migration was analyzed using the Boyden chamber assay. Migrating cells were fixed with methanol, stained with Giemsa, and lysed. Absorbance was measured at a wavelength of 560 nm. Values represent the mean±standard deviation of three independently performed experiments (**P<0.01; two-way Mann-Whitney)