Figure 2. Selective detachment of HK II from mitochondria induces PTP opening and cell death.

(A) HK II detachment from mitochondria of HeLa cells following a 1-hour treatment with TAT-HK (20 µM). Western immunoblots of a mitochondrial and of a cytosolic fraction show a redistribution of both HK II and cytochrome c into the cytosol. Blots were also probed with prohibitin as a mitochondrial marker and with actin as a cytosolic marker to check for fraction purity. (B) HK II immunoprecipitation in HeLa cells kept in control conditions or treated for 1 hour with Debio 025 (DB, 8 µM), TAT-HK (20 µM), or both. Co-immunoprecipitation of VDAC is shown. (C) Output of multiparametric FACS analyses show apoptosis induction of HeLa cells exposed for 2 hours to a control peptide linked to the TAT sequence (TAT-Ctr, 40 µM) or to the reported concentrations of TAT-HK with or without pre-incubation with Debio 025 (DB, 8 µM). Diagrams and percentages of the different cell populations are as in Fig. 1B. (D) PTP opening of digitonized HeLa cells measured with the Ca²⁺ retention capacity assay. Experiments were started by the addition of cells (not shown) followed by pulses of the indicated concentrations of Ca²⁺; where indicated, TAT-HK (30 µM) was added to cells 45 minutes before permeabilization with digitonin. TAT-Ctr (40 µM) did not change the number of Ca²⁺ pulses (not shown). Calcium Green-5N fluorescence is reported as arbitrary units on the y axis. (E) MPT susceptibility to ROS (t_MPT) in intact cardiac myocytes is significantly enhanced by TAT-HK while the negative control peptide TAT-Ctr has no effect. t_MPT measurements were performed after 1 hr treatment with peptides. Figure represents four independent experiments with 50–60 cells per groups examined. (F) Respiration assay performed on mouse liver mitochondria. The rate of oxygen consumption (ng AtO stands for ngatoms of oxygen) is plotted vs. different concentrations of the HK peptide. The rate of respiration is displayed in basal conditions (▪), after ADP administration (•) or after dinitrophenol administration (▴). All reported measures in the Figure are representative of at least four experiments.