Figure 1.
Kinetic of clofibrate-induced death in Jurkat cells.
Morphological appearance (A: phase contrast microscopy, B: DAPI staining) of Jurkat cells exposed to clofibrate (CF) 1 mM for 45 min (Co: control). (C) percentages of cells with hypodiploid DNA content (apoptotic; see Penna et al., 2009) after exposure to CF (concentration on top of the bars, time on X axis). Data are expressed as mean ± SD (n = 3). ** p<0,01 vs Co; *** p<0,001 vs Co.
Figure 2.
Mitochondrial involvement in clofibrate-induced apoptosis.
Shift of the FL2 (590 nm)/FL1 (529 nm; green to red) fluorescence ratio of JC-1 in cells treated with clofibrate (CF) for 5 min (A: control (C); B: CF). (C) representative western blot showing the time course of Smac/DIABLO and cytochrome c appearance in the cytosolic fraction (surnatant, SN). Cytochrome c oxidase subunit IV (COX4) serves as loading control for mitochondrial enriched fraction (insoluble, INS) and actin for cytosolic fraction. (D) western blotting pattern representative of cyclosporine A and zVAD pretreatment on cytochrome c release in cells exposed to CF for 30 min.
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 ± SD, n = 3) are expressed as fold increase respect to Co (control; black bar); ** p<0,01 vs Co; *** p<0,001 vs Co; § p<0,05 vs CF; §§ p<0,01 vs CF; §§§ p<0,001 vs CF.
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 ± SD (n = 3). ** p<0,01 vs Co; *** p<0,001 vs Co; §§ p<0,01 vs CF; §§§ p<0,001 vs CF.
Figure 5.
Phosphorylation of eIF2α and JNK activation in clofibrate-treated cells.
(A) Representative western blottings of phosphorylated eIF2α 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 ± SD (n = 3). ** p<0,01 vs Co; *** p<0,001 vs Co; § p<0,05 vs CF.
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 ± SD (n = 3). ** p<0,01 vs Co; *** p<0,001 vs Co; §§§ p<0,001 vs CF.
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 µM) and analysed after 45min. Data are expressed as mean ± SD (n = 3). *** p<0,001 vs Co; §§§ 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 ± 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; §§ 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).