Figure 3. MCF-7 cells are killed by transient expression of BimEL or Bid, are protected by stably expressed Bcl-XL or Bcl-2 and depend on MCL-1 for survival.

(A) Transient expression of exogenous ^VtBid (blue) and ^VBimEL (red) killed MCF-7 cells. MCF-7 cells were largely resistant to expression of ^VBad (green) as the resulting cell death was similar to expression of the control protein Venus (black). Images of individual cells were assessed for apoptosis based on staining with TMRE and the nuclear dye DRAQ five using a multiparametric linear classifier. An increase in the percentage of cells scored as dead or dying (% Dead) as a function of Venus intensity demonstrated that the ^VtBid and ^VBimEL fusion proteins retain pro-apoptotic activity. Error bars indicate standard error for three independent replicates. At least 30 cells were analyzed at each point representing a Venus intensity bin. (B) Immunoblotting of lysates from MCF-7 cells (lane 1) and MCF-7 cells expressing exogenous ^CBcl-XL (lane 2) or Bcl-XL (Lane 3) with an antibody to Bcl-XL demonstrated that the exogenous proteins are at least 20-fold over-expressed compared to endogenous Bcl-XL. The same blot was probed for β-actin as a loading control. (C) Cells classified as dead or dying (% Dead) for 3 cell lines (MCF-7 and MCF-7 expressing either ^CBcl-XL or ^CBcl-2), treated with 2 µg/ml cyclohexamide (CHX), or CHX plus TNFα (250 ng/ml and 500 ng/ml) for 24 hr. Data (% Dead) for three independent replicates (circles) and the mean of the replicates (line) are plotted. A one-way ANOVA test was performed with a Dunnett's Multiple Comparison post-test (Graphpad Prism), to compare all treated wells with untreated controls for each cell line. (D) Overexpression of ^CBcl-2 and ^CBcl-XL protected cells from BH3-mimetics. Points represent the average percentage of cells classified as dead or dying (% Dead) for individual replicates, with the mean of the replicates indicated by a line. MCF-7 cells and MCF-7 cells expressing either Bcl-2 or Bcl-XL were treated with 1.25 µM (dot) and 20 µM (circle) ABT-199, A-1331852 or ABT-263 as indicated above. Neither selective nor dual inhibition of Bcl-2 and Bcl-XL induced substantial cell death in MCF-7 cell lines (all below 20% Dead) demonstrating these cells are not highly dependent on expression of either of these anti-apoptotic proteins. Nevertheless expression of Bcl-2 or Bcl-XL reduced cell death to barely detectable levels. (E) The MCL-1 inhibitor, S-63845 (Servier) kills MCF-7 (black), but does not kill MCF-7 ^CBcl-XL (blue) and MCF-7 ^CBcl-2 (red) cells. An EC50 value of 530 ± 6 nM, was calculated for MCF-7 cells in GraphPad Prism using a non-linear fit of normalized data on a log scale (log(agonist) verses response, and variable slope (four parameters)).