Figure 3.

AUL12 sensitizes the PTP to opening in a ROS-dependent way. (a) PTP opening of SAOS-2 cells treated with AUL12 is measured with the whole-cell CRC assay. Fluorescence of Calcium Green-5N in digitonin-permeabilized cells is reported as arbitrary units on the y axis. As the probe does not permeate mitochondria, Ca²⁺ uptake into the organelles after each pulse (5 μM Ca²⁺) is displayed by a rapid decrease of the fluorescence spike. Pore inhibitors and inducers are expected to increase or decrease, respectively, the number of spikes before permeability transition, that is, a sudden and marked fluorescence increase, occurs. AUL12 was added to cells at the indicated concentrations 3 h before permeabilization with digitonin. Where indicated, cyclosporine A (CsA) was added 5 min before starting the assay. The bar graphs in (b) report the total Ca²⁺ uptake before PTP opening (results are mean±S.D.; n=6). (c, e) Representative CRC experiments performed on isolated liver mitochondria treated with different concentrations of AUL12 (c) and/or with N-acetyl-cysteine (NAC, 1 mM; (e)). The bar graphs in (d, f) report the total Ca²⁺ uptake before PTP opening (results are mean±S.D.; n=6). Statistical significance was measured with a Student's t test and is indicated by asterisks (**P<0.005)