Rapid Jurkat Apoptosis by Clofibrate Figure 5. Phosphorylation of eIF2a and JNK activation in clofibrate-treated cells. (A) Representative western blottings of phosphorylated eIF2a and JNK (46 and 54 kDa isoforms). (B) histograms refer to the densitometric analysis. Data are expressed as arbitrary units. (C) Cells were pretreated with the JNK inhibitor SP600125, then exposed to clofibrate (CF) and analysed after 45min. Histograms refer to percentages of cells with hypodiploid DNA content (black bars: untreated; grey bars: CF-treated). Data are expressed as mean 6 SD (n = 3). ** p,0,01 vs Co; *** p,0,001 vs Co; 1 p,0,05 vs CF. doi:10.1371/journal.pone.0045327.g005 its cytotoxic activity also on normal cells, primary T lymphocytes were exposed to the drug. The results shown in Fig. S2A demonstrate that 45 minutes exposure to 1 mM clofibrate slightly modifies the precentage of hypodiploid cells observed in control cultures. The results do not change when T cells are stimulated with anti-CD3 antibodies for 36 h, in order to have them in a proliferative condition (% of hypodiploid cells: C = 11, CF = 13). In contrast to what observed in Jurkat cells (Fig. 1C), exposure of T lymphocytes to low CF concentrations (0.5 mM) for a period longer than the standard experimental schedule (from 459 to 8 h) markedly delays the onset of cell death, leading to slightly increased percentage of hypodiploid cells only after 8 h treatment (Fig. S2B). Mitochondrial Alterations Induced by CF A drop in the mitochondrial membrane potential (Fig. 2A–B) as well as a substantial release of cytochrome c to the cytosol (Fig. 2C) manifest rapidly and are already remarkable after a 5 min exposure of Jurkat cells to CF. By contrast, Smac/Diablo is barely detectable in the cytosol (Fig. 2C). The release of cytochrome c to the cytosol is clearly attenuated when the opening of the permeability transition pore is inhibited with cyclosporine A (Fig. 2D) or when cells are pretreated with the polycaspase inhibitor zVAD (Fig. 2D). The latter finding indicates that cytochrome c release is, at least in part, an effect rather then a cause of a proteolytic event inhibited by zVAD. CF-induced Death is Caspase-dependent In a next step, Jurkat cells have been assayed for the enzymatic activity of an effector (3) and three initiator (2, 8 and 9) caspases. As Fig. 3A illustrates, caspase 2-like activity rapidly increases in the first time interval (15 min) and is maintained active afterwards. Conversely, the increase of both caspase 8- and 9-like activities is more evident in the late phases (30–45 min). Finally, the caspase 3- like activity markedly increases in a virtually linear fashion over the whole 45 min interval. Caspase 2 and 3 activation is confirmed by western blotting analysis: the precursor isoforms progressively decrease after exposure to CF, while processed (active) caspase 3 becomes detectable (Fig. 3B), in parallel with an early and extensive cleavage of its endogenous substrate PARP (Fig. 3B). Jurkat cells lack any detectable caspase 4 expression (our original observation, not shown) as well as any caspase 12 activity, which is a general property of human cells [29]. Incidentally, no change in the level of procaspase 12-like immunoreactive protein occurs in CF-treated Jurkat cells over the whole experimental time (Fig. 3B). CF-induced caspase 3 activation is completely abrogated when cells are pretreated with either polycaspase (zVAD) or caspase 3 (DEVD) inhibitors (Fig. 3C). Among the initiator caspases, only caspase 2 inhibition with VDVAD partially prevents caspase 3 activation, while inhibitors of caspase 8 (IETD) or 9 (LEHD) are ineffective. As emerging from inhibitor tests, CF-induced cell death is caspase-dependent (Fig. 4A). Cells treated with zVAD become PLOS ONE | www.plosone.org 6 September 2012 | Volume 7 | Issue 9 | e45327