Figure 3.

The yeast BH3 domain induces apoptotic changes in isolated mitochondria as well as in mammalian and yeast cells. (A) Amino-acid sequence alignment of the native yeast BH3 domain and the BH3^D295K and BH3^L290K point mutants. (B) Immunoblot analysis of WT yeast cells for FLAG-tagged Ybh3p, Ybh3p^ΔBH3, Ybh3p^D295K or Ybh3p^L290K. (C, D) Death determined by clonogenicity (C) and quantification of ROS accumulation using DHE → Eth conversion (D) of yeast cells overexpressing Ybh3p, Ybh3p^ΔBH3, Ybh3p^D295K or Ybh3p^L290K after treatment with 120 mM acetic acid for 1 or 4 h. Death rates were calculated by setting the survival of vector control cells to 100% and thus 0% death (mean±s.e.m., n=8). (E–H) Swelling of mouse liver mitochondria as indicated by loss of absorbance (ABS) at 490 nm. Mitochondria were pre-incubated or not with 5 μM CsA, followed by the administration of 2.5 μM (E) or 5 μM (F) of native (BH3) or mutated (BH3^L290K) peptide or indicated concentrations of Ca²⁺(G). To permit direct comparison, swelling after 14 min under all conditions analysed was depicted in (H). Representative experiments are shown, with data representing mean±s.e.m. of three replicates. (I, J) Immunoblot analysis of supernatants and mitochondrial pellets (I) from mouse liver mitochondria incubated with the indicated concentration of native (BH3) or mutated (BH3^L290K) peptide using an antibody specific for cyt c. For supernatants, the signal intensities of cyt c were quantified and normalized to VDAC intensities of deployed mitochondria (J) (mean±s.e.m., n=3). (K) Non-small cell lung cancer (A549) cells were incubated with 30 μM of the native (BH3) or mutated (BH3^L290K) peptide, followed by the flow cytometric assessment of Δψ_m dissipation (DiOC₆(3)^low) and loss of plasma membrane integrity (PI+) (mean±s.e.m., n=5). (L) A549 cells transfected with a control siRNA (siUNR) or with siRNA targeting BAX, BAK or with a combination of these were treated with 30 μM of the native (BH3) or mutated (BH3^L290K) peptide followed by the flow cytometric assessment of Δψ_m dissipation (DiOC₆(3)^low) and loss of plasma membrane integrity (PI+). Data represent mean±s.e.m. and one representative experiment is shown with n=3. ^*P<0.05, ^**P<0.01 and ^***P<0.001.