Rapid Jurkat Apoptosis by Clofibrate Figure 7. Effect of Ca2+ influx on clofibrate-induced apoptosis. (A) Cells were pretreated with OG-Bapta-AM or EGTA, then exposed to clofibrate (CF) and/or the Ca2+ ionophor A23187 (5 mM) and analysed after 45min. Data are expressed as mean 6 SD (n = 3). *** p,0,001 vs Co; 111 p,0,001 vs CF. Histograms refer to percentages of cells with hypodiploid DNA content (black bars: without CF; grey bars: CF-treated). (B) cells were incubated with OG-Bapta1-AM and treated with CF and/or A23187. Intracellular calcium release was measured by the increase of 523 nM fluorescence. (C) Caspase 3 enzymatic activity in cells treated with CF and/or A23187 for 30 min. Data (means 6 SD, n = 3) are expressed as fold increase respect to control. * p,0,05 vs Co; ** p,0,01 vs Co; *** p,0,001 vs Co; 11 p,0,01 vs CF. (D) western blotting representative of the effect of CF and/or A23187 for 30 min on cytochrome c release. Cytochrome c oxidase subunit IV (COX)4 serves as loading control for mitochondrial enriched fraction (P) and actin for cytosolic fraction (SN). doi:10.1371/journal.pone.0045327.g007 prominent at 5 min and virtually complete at 15 min, while a 120 kDa fragment appears after 30 min (Fig. 6A). The 120 kDa fragment is generated by caspase 3 only, whereas both calpain and caspase 3 can cut a similar though not identical 150 kDa fragment [33]. We thus used appropriate inhibitors in order to discriminate between the latter two possibilities. As Fig. 6A illustrates, the 150 and 120 kDa products of fodrin cleavage both nearly disappear in the presence of zVAD, whereas the calpain inhibitor calpeptin affords no more than a weak decrease of the 150 kDa fragment. In apparent contrast with the latter finding, calpeptin markedly protects Jurkat cells from death by CF, yet such protection in not provided at all by another calpain inhibitor such as PD150606 (Fig. 6B). Alto- gether, these observations suggest that calpains make no significant contribution to CF-induced apoptosis in Jurkat cells. Ca2+ Influx Prevents CF-induced Apoptosis Finally, we tested the hypothesis that altered Ca2+ homeo- stasis could be critical for CF cytotoxicity, even if not necessarily through calpain activation. CF was previously shown [35] to induce transient increases of the [Ca2+]i in Jurkat cells. The relevance of such increase to CF-induced apoptosis was not investigated, however. In the present work, neither intracellular nor extracellular Ca2+ chelators, such as OG-BAPTA-AM and EGTA, respectively, prevent CF-induced apoptosis (Fig. 7A). In addition, the Ca2+ ionophore A23187 by itself exerts no appreciable toxicity on Jurkat cells, at least over the 45 min interval herewith adopted (Fig. 7A). This observation implyes that any change of [Ca2+]i caused by CF or the Ca2+ influx induced by A23187 separately are well tolerated by these cells. Surprisingly, however, CF cytotoxicity is fully antagonized by A23187 and such protection is abrogated when cells are in the presence of EGTA (Fig. 7A). Not only enforced Ca2+ influx is required for cell survival, but also other CF effects such as caspase 3 activation and cytochrome c release are reduced by A23187 (Fig. 7C–D). In agreement with [35], we found CF to induce an early increase of [Ca2+]i, peaking at 1 min and followed by a progressive decline (Fig. 7B). A23187 causes a prompt [Ca2+]i rise that is largely maintained till the end of the assay. Finally, cotreatment with CF and A23187 results in a complex pattern including an initial [Ca2+]i spike followed by a decline to a suprabasal plateau and by an eventual rise to PLOS ONE | www.plosone.org 8 September 2012 | Volume 7 | Issue 9 | e45327