Rapid Jurkat Apoptosis by Clofibrate Figure 4. Effect of different caspase inhibitors and caspase 2 silencing on apoptotic death in cells exposed to clofibrate. (A) cells were pretreated with the indicated inhibitors, exposed to clofibrate (CF) and analysed after 45 min. Histograms refer to percentages of cells with hypodiploid DNA content (black bars: untreated; grey bars: CF-treated). (B) sh-RNA effectively abrogates caspase 2 expression in Jurkat cells. Co = control, Scr = scramble shRNA, shCSP2 = caspase 2-specific shRNA; Lane 1: molecular weight marker. Tubulin is used as loading control. (C) flow- cytometric analysis showing the percentages of cells with hypodiploid DNA content of the above mentioned cells exposed to 1 mM clofibrate for 45 min. Data are expressed as mean 6 SD (n = 3). ** p,0,01 vs Co; *** p,0,001 vs Co; 11 p,0,01 vs CF; 111 p,0,001 vs CF. doi:10.1371/journal.pone.0045327.g004 analyzed using a specific software (TotalLab, NonLinear Dynam- ics, Newcastle upon Tyne, UK). Significance of the differences was evaluated by the Student’s ‘t’ test. Supplemental Material Primary T cells were isolated using buffy coats from healthy individuals (kindly supplied by CeRMS, Centro Ricerche Medicina Sperimentale, Torino, Italy). PBMCs were separated using the Ficoll-Paque reagent (GE Healthcare, Milano, Italy) following the manufacturer’s instruction. Pan T cells were obtained by negative selection retaining the non-target cells in the magnetic field of a MACS Column (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) after incubation with a cocktail of biotin-conjugated monoclonal anti-human antibodies against CD14, CD15, CD16, CD19, CD34, CD36, CD56, CD123, and CD235a. The extracted cells were rapidly seeded in complete medium (same as Jurkat cells) and immediately exposed to CF for cytotoxicity assays. Results When exposed to 1 mM CF for 45 min, the standard treatment schedule adopted throughout the present work, approximately 50% of Jurkat cells develop a frankly apoptotic morphology characterized by cell shrinkage due to nuclear and cytoplasmic condensation with nuclear fragmentation (Fig. 1A–B). Compara- ble effects have been observed on different cell lines, such as human HL-60 promyelocytic leukemia (Fig. S1A) or human CCRF-CEM acute lymphoblastic leukemia (data not shwn), and the murine Colon-26 carcinoma cells (Fig. S1B). Noteworthily, a time/dose study shows that apoptosis is also induced by CF at concentrations lower than 1 mM (Fig. 1C), even if propagating more slowly in the cell population. These concentrations are well within the usual therapeutic (antilipidemic) range that approx- imates 0.5 mM in patient blood [28]. More pertinently, in pharmacological terms, optimal therapeutic plasma levels of CF have been reported to be around 0.5 mM ([28]; MICROMEDEX Healthcare Series, POISINDEX Managements, Clofibrate and related agents, www.thomsonhc.com). To verify if clofibrate exerts PLOS ONE | www.plosone.org 5 September 2012 | Volume 7 | Issue 9 | e45327