Rapid Jurkat Apoptosis by Clofibrate Figure 6. Fodrin cleavage and effect of calpain inhibition in clofibrate-treated cells. (A) Representative western blotting of fodrin cleavage in clofibrate (CF)-exposed Jurkat cells in the presence or in the absence of zVAD and calpeptin. (B) Cells were pretreated with the calpain inhibitors calpeptin or PD150606, then exposed to 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; 111 p,0,001 vs CF. doi:10.1371/journal.pone.0045327.g006 almost completely refractory to CF cytotoxicity. Caspase 2 and 3 inhibitors (respectively, VDVAD and DEVD) afford no more than a partial protection, which becomes higher when they are used concomitantly. Finally, inhibitors such as IETD (caspase 8) or LEHD (caspase 9) do not rescue cells from apoptosis, which is consistent with their inability to affect caspase 3 activation (Fig. 3B). In keeping with this finding, cyclosporine A, which is known to suppress mitochondrial membrane permeability transitions, af- fords no protection from cell death (Fig. 4A). To clarify if caspase 2 activation is crucial to CF citotoxicity, its expression in Jurkat cells was abrogated by means of shRNA (Fig. 4B). The lack of caspase 2 rendered Jurkat cells more, although not completely, resistant to CF-induced apoptosis. Indeed, the percentage of hypodiploid cells was more than 50% in wild-type cultures, and about 30% in caspase 2-depleted cells (Fig. 4C). CF Induces ER Stress At present, caspase 2 is not univocally associated with a precise cell death pathway, yet there is evidence that on ER stress it may act upstream of mitochondria to promote cytochrome c release [30,31]. Therefore, we assessed the occurrence of ER stress in CF- treated Jurkat cells by examining the phosphorylation status of the stress-activated SAPK/JNK (46 and 54 kDa isoforms) and of the translation inhibitory factor eIF2a and in both cases observed its progressive increase (Fig. 5A–B). Furthermore, apoptosis by CF is partially prevented in cultures pretreated with the JNK inhibitor SP600125, which suggests a role for JNK in this death process (Fig. 5C). CF-induced Apoptosis does not Rely on Calpain Activation ER stress is often associated with or elicited by Ca2+ mobilization from ER stores, which results in increased cytosolic Ca2+ concentrations ([Ca2+]i) that may trigger activation of the Ca2+-dependent calpain system [32]. A physiological substrate of the latter is fodrin, a cortical cytoskeletal protein, whose cleavage can thus denote an ongoing calpain activation [33] and has implications for membrane blebbing and phosphati- dylserine externalization during apoptosis [34]. In CF-treated Jurkat cells, fodrin cleavage into a 150 kDa product is already PLOS ONE | www.plosone.org 7 September 2012 | Volume 7 | Issue 9 | e45327