Rapid Jurkat Apoptosis by Clofibrate Figure 3. Caspase activation in clofibrate-treated cells. (A) Kinetic of caspase enzymatic activity evaluated by measuring the cleavage of specific fluorogenic substrates. Data are expressed as fold increase respect to control. (B) representative western blotting patterns of caspase and PARP (poly-ADP-ribose polymerase) expression in CF-treated Jurkat cells. Tubulin serves as loading control. (C) Caspase 3 enzymatic activity in cells pretreated with the indicated inhibitors before CF (clofibrate; grey bars) exposure for 30 min. Data (means 6 SD, n = 3) are expressed as fold increase respect to Co (control; black bar); ** p,0,01 vs Co; *** p,0,001 vs Co; 1 p,0,05 vs CF; 11 p,0,01 vs CF; 111 p,0,001 vs CF. doi:10.1371/journal.pone.0045327.g003 (VDVAD-AMC (AMC:7-amino-4-methylcoumarin)), DEVD- AMC, IETD-AMC, LEHD-AMC; Biomol, Plymouth Meeting, PA, USA). The reaction was stopped with 0.1% trichloroacetic acid, and the fluorescence read in a Perkin-Elmer fluorometer (excitation 380 nm – emission 460 nm). Free AMC was used as working standard. Western Blotting Cells were suspended in 0.25% sodium deoxycholate and homogenized by sonication. For cytochrome c detection, cells were suspended in 20 mM HEPES-KOH, pH 7.2, containing 250 mM sucrose, 1 mM EDTA, 0.025% digitonin and freshly added 0.1 mM PMSF, then centrifuged (14,000 g, 30 min, 4uC) to obtain the mitochondrial (pellet) and the cytosolic (supernatant) fractions. Protein concentration was determined by the method of Lowry et al. (1952), using BSA as working standard. Equal amounts of protein (30 mg) were heat-denaturated in sample-loading buffer (50 mM TRIS-HCl, pH 6.8, 100 mM DTT, 2% SDS, 0.1% bromophenol blue, 10% glycerol), resolved by SDS-PAGE and transferred for 2 h to nitrocellulose membranes (Bio-Rad, Hercules, CA, USA). Protein transfer was checked by Ponceau S staining. The filters were then blocked with Tris-buffered saline (TBS) containing 0.05% Tween and 5% non-fat dry milk, and incubated overnight with the following primary antibodies: Smac/ DIABLO, poly-ADP-ribose polymerase (PARP), caspase 2, caspase 3, caspase 12, fodrin, JNK (Santa Cruz Biotechnology, Santa Cruz, CA, USA); cytochrome c oxidase subunit IV (COX4), p-eIF2a, eIF2a, p-JNK (Cell Signaling, Danvers, MA, USA); cytochrome c (Becton & Dickinson, Mountain View, CA, USA); actin and tubulin (Sigma, St. Louis, MO, USA). Peroxidase- conjugated IgG (Bio-Rad, Hercules, CA, USA) was used as secondary antibody. The membrane-bound immune complexes were detected by an enhanced chemiluminescence system (Santa Cruz Biotechnology, Santa Cruz, CA, USA) on a photon-sensitive film (Hyperfilm ECL; GE Healthcare, Milano, Italy). Bands were quantified by densitometric scanning of the films and elaborated as described in ‘Data analysis and presentation’. Caspase-2 Silencing The shRNA lentiviral vector pLKO.1,-puro bearing the hairpin sequence CCGGGTTGAGCTGTGACTAC- GACTTCTCGAGAAGTCGTAGTCACAGCT- CAACTTTTTG was transfected in packaging HEK 293T cells using calcium phosphate. 24 hours later, the supernatant contain- ing the lentiviral particles was filtered (0,22 mm) and 1 ml added to 106 Jurkat cells. Infected cells were selected adding puromycin 1mg/ml during 2 weeks, tested for silencing efficiency and used for subsequent experiments. Data Analysis and Presentation Results are expressed as means 6 standard deviation (SD). Each experiment was performed in triplicate or repeated three times. Image quantification was obtained by densitometry and bands PLOS ONE | www.plosone.org 4 September 2012 | Volume 7 | Issue 9 | e45327