Rapid Jurkat Apoptosis by Clofibrate Figure 2. Mitochondrial involvement in clofibrate-induced apoptosis. Shift of the FL2 (590 nm)/FL1 (529 nm; green to red) fluorescence ratio of JC-1 in cells treated with clofibrate (CF) for 5 min (A: control (C); B: CF). (C) representative western blot showing the time course of Smac/ DIABLO and cytochrome c appearance in the cytosolic fraction (surnatant, SN). Cytochrome c oxidase subunit IV (COX4) serves as loading control for mitochondrial enriched fraction (insoluble, INS) and actin for cytosolic fraction. (D) western blotting pattern representative of cyclosporine A and zVAD pretreatment on cytochrome c release in cells exposed to CF for 30 min. doi:10.1371/journal.pone.0045327.g002 70% ethanol for at least 30 min, incubated at room temperature in PBS containing DNase-free RNase (Type II-A) and propidium iodide at the final concentrations of 0.4 mg/ml and 10 mg/ml, respectively. Cells were then analyzed with a FACScan flow cytometer (Becton & Dickinson, Mountain View, CA, USA) equipped with a 488 nm argon laser and three filters, respectively transmitting at 530 nm (FL1), 585 nm (FL2) and above 620 nm (FL3). Data were analysed with the CellQuest software (Becton & Dickinson). The percentage of apoptotic events has been assessed by evaluating the accumulation of cells characterized by a ,2n DNA fluorescence. Mitochondrial depolarization was detected on unfixed cells measuring the fluorescence emission shift of the lipophilic cationic probe 5,5-6,6-tetra-chloro-1,1-3,3-tetraethylbenzimidazolyl-carbo- cyanine iodide (JC-1, Molecular Probes, Invitrogen Corporation, Carlsbad, CA, USA). JC-1 exhibits potential-dependent accumu- lation in mitochondria, indicated by a fluorescence emission shift from green (529 nm) to red (590 nm). The loss of mitochondrial membrane potential is indicated by a decreased ratio between red and green fluorescence [27]. Intracellular Ca2+ fluctuations were measured using the calcium indicator OregonGreen488-Bapta1-AM (Molecular Probes, Invi- trogen Corporation, Carlsbad, CA, USA). Briefly, cells (2?106) were loaded with the specific calcium probe (2 mM, 30 min, room temperature) in complete medium without phenol red, centrifuged (600 g, 5 min) and resuspended in 1 ml of the same medium. Data capture was performed recording the FL1 mean on 10,000 cells for each time point. CF and/or A23187 were added after reading the baseline fluorescence. Caspase Activities Cells were resuspended in 20 mM HEPES-KOH, pH 7.5, containing 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 1 mM PMSF, frozen and thawed, sonicated, centrifuged (14,000 g, 15 min, 4uC), and the supernatant collected. Aliquots corresponding to 20 mg protein were diluted in caspase buffer (25 mM HEPES, pH 7.5, 0.1% CHAPS, 10% sucrose, 10 mM DTT) and assayed for caspase 2, 3, 8, and 9 activities by 1 h incubation at 37uC in the presence of 20 mM substrates PLOS ONE | www.plosone.org 3 September 2012 | Volume 7 | Issue 9 | e45327