Fig. 6. ATAP induces the loss of mitochondrial membrane potential and perturbs membrane permeability in planar lipid bilayers. (A) Effect of GFP-ATAP on the mitochondrial membrane potential. Mitochondrial outer membrane potential was observed using MitoTracker Red (CM-H₂ TMRos) dye. HeLa cells were transfected with GFP-ATAP, GFP-mHR7 or GFP-TAxL and cultured in the presence of 50 µM OPH. 24 hours after transfection, cells were incubated in medium containing 50 nM of MitoTracker for 2 hours and observed under a fluorescence microscope. Bar, 10 µm. (B) Green (GFP) and red (MitoTracker) double-positive cells were quantified from about 300 GFP-positive cells from three different fields. Data are expressed as the mean ± s.e. (C) Effect of ATAP on cytochrome c release. Left panel, HEK293 cells were transfected with 1 µg of pEGFP (lane 1), pEGFP-ATAP (lane 2) or pEGFP-mHR7 (lane 3). 20 µg mitochondria-free cytosol proteins were analyzed by western blotting using anti-cytochrome c antibody. Right panel, mitochondria membrane isolated from BMK-D3 cells were incubated with synthetic ATAP and mHR7 peptides (100 µM). ATAP induced cytochrome c release into the supernatant (S), whereas cytochrome c remained in the pellet (P) in preparations treated with mHR7 and DMSO (as a control). (D) Effect of synthetic ATAP and mHR7 peptides on the membrane permeability of planar lipid bilayer. The peptides (11.1 µM) were added to the cis chamber. Current traces at the corresponding holding potentials were measured from recording solution of 200 mM KCl (cis) and 50 mM NaCl (trans). Data are representative of n=5 for ATAP and n>35 for mHR7.