Fig. 4: Effect of TRAP1 interaction on F-ATP synthase enzymatic activity and PTP opening.

A Oligomycin-sensitive ATPase activity on human U87 glioblastoma cells with or without TRAP1 (SCR and KO, respectively). Where indicated, the TRAP1 inhibitor i5 was used at 25 µM. The histogram reports the mean ± SEM of 4 independent experiments. B BN-PAGE followed by in gel ATPase activity (top left) on U87 mitochondria extracted with the indicated concentrations of digitonin. Quantification of ATPase activity is reported as the mean dimer/monomer ratio from three independent measurements; *p < 0.05, **p < 0.01 ***p < 0.005 (bottom, left). BN-PAGE followed by Western immunoblotting (top, right) was probed with antibodies for F-ATP synthase subunit β to detect enzyme monomers, dimers and oligomers; anti-SDHA antibody was used as a loading control. C ATPase activity measured in submitochondrial particles (SMPs) from pig heart pre-incubated with 1 nmol of TRAP1 or CyPD per mg of protein alone or sequentially for 5 min at 37 °C. Where indicated, the TRAP1 inhibitor i5 (100 μM) or CsA (4 μM) were preincubated for 5 min at 37 °C. Data are shown as percentage changes with respect to untreated SMPs. D Calcein cobalt assay of PTP opening on U87 cells. Cells were stained with calcein AM at time zero; 2 μM Ca²⁺ ionophore A23187 was added where indicated in the absence or in the presence of 2 μM CsA. Image frames were collected at 30-s intervals and fluorescence values (mean ± SEM n s = 4) were quantified. E Mitochondrial membrane potential assessment in U87 cells after the addition of 2 µM cl-HK2pep. Cells were stained with TMRM probe (20 nM + 1 µM CsH); data are reported as mean ± SD; **p < 0.01 with a TwoWay Anova p < 0,001, Bonferroni post-test analysis.