Figure 1
CD95 induces a BH3-dependent calcium signal that promotes TNBC cell motility. (a) MDA-MB-231 and BT549 cells were pre-incubated for 1 h in the presence of 0.1 and 10 μM ABT-737, respectively, and then treated or untreated with cl-CD95L (100 ng/ml) for 24 h. 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<0.001; two-way Mann-Whitney). Bars=50 μm. (b) MDA-MB-231 and BT549 cells were pre-incubated for 1 h with or without non-toxic doses of ABT-737 (0.1 and 10 μM, respectively) and then treated or untreated with cl-CD95L (100 ng/ml) for the indicated amount of time. Cells were lysed and immunoblotting was performed as indicated. Akt was used as a loading control. (c) MDA-MB-231 and BT549 cells were pre-incubated for 1 h in the presence or absence of ABT-737 (0.1 μM). Cells were loaded with the Ca2+ probe FuraPE3 and then stimulated with cl-CD95L (100 ng/ml). The cytosolic calcium concentration was monitored via the ratio F/F0 (relative Ca2+[CYT]). Data represent the mean±s.e. of the mean of F/F0 (n>50 cells, at least three independent experiments). (d) MDA-MB-231 cells were infected with lentiviruses encoding shRNAs targeting different regions of Bclx mRNA. After puromycin selection, cells were lysed and the amounts of BclxL and Bcl-2 were evaluated by immunoblotting. β-Actin served as a loading control. (e) Migration of cells depicted in (d) was assessed using the Boyden chamber assay in the presence or absence of cl-CD95L (100 ng/ml) for 24 h. Migrating cells were fixed, stained (Giemsa), and lysed. Absorbance of migrating cells 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). (f) MDA-MB-231 cells were transduced with lentiviruses encoding shRNAs targeting different regions of Bcl-2. After puromycin selection, cells were lysed and the expression levels of Bcl-2 and BclxL were evaluated by immunoblotting. β-Actin served as a loading control. (g) Migration of cells depicted in (f) was evaluated by the Boyden chamber assay in the presence or absence of cl-CD95L (100 ng/ml) for 24 h. Migrating cells were fixed, stained (Giemsa), and lysed. Absorbance of migrating cells was measured at a wavelength of 560 nm. Values represent the mean±s.e. of the mean of three independently performed experiments (**P<0.01; two-way Mann-Whitney). (h) The calcium response was assessed in MDA-MB-231 cells infected with lentiviruses encoding various shRNAs targeting BclxL (left panel) or Bcl-2 (right panel). Cells were loaded with the Ca2+ probe Fluo2 and then stimulated with cl-CD95L (100 ng/ml). The cytosolic calcium concentration was monitored via the ratio F/F0 (relative Ca2+[CYT]). Data represent the mean±s.e. of the mean of F/F0 measured in GFP-positive cells (n>50 cells, at least three independent experiments)
