TSA generates ROS and activates the caspase-dependent cell death pathway. (A) Effect of cycloheximide and actinomycin-D on TSA-induced apoptosis. CD4+ T-cells were pre-treated with either CHX or act-D for 1 h after which TSA was added to the culture medium at a final concentration of 100 nM. After 20 h, levels of apoptosis were assessed by PI staining. (B) Western blots showing the effect of TSA on levels of caspase-3 and -10. CD4+ T-cells were cultured for 20 h in the presence of medium containing growing concentrations of TSA or DMSO alone (-TSA). After 20 hours the cells were harvested and cell extracts prepared as described in the Materials and Methods section. Western blots were prepared using one of the following antibodies: caspase-3 (H-277), or caspase-10 p20 (H-131) from Santa Cruz Biotechnology, Inc. (USA). Reversible staining of blots with Ponceau S was used as loading and blotting control. A typical blot is shown. (C) Effect of TSA on cell death receptor expression. Spleen cells from C57BL/6 mice were cultured for 48 h in the presence of soluble anti-CD3 antibody (0.5 μg/ml), whereafter CD4+ T-cells were isolated by magnetic cell separation as described (materials and methods section). Cells were subsequently stimulated with immobilized anti-CD3 mAb or cultured in the presence of IL-2 (10 U/ml), harvested after 20 hours in the presence of various concentrations of TSA, and the levels of CD95 and CD95L analysed by flow cytometry. Inset in the upper right quadrant of each plot are the percentage of cells that gated to the various quadrants (UL – upper left; UR – upper right; LL – lower left; and LR – lower right). Logic quadrants were placed such that the cells in a non-stimulated sample gate to the LL quadrant. Only live cells are included in the analysis. (D) TSA induces ROS generation. CD4+ T-cells were incubated for 24 h in the presence of medium containing IL-2 (10 U/ml), or anti-CD3 coupled beads (2:1 beads to cells ratio) after which either TSA (final concentration of 10 nM; bold black open histogram) or DMSO alone (black open histogram) was added. After 20 hours the cells were analysed for DHE and DCFDA fluorescence. (E) TSA-induced T-cell apoptosis can be inhibited by free radical scavengers and mitochondrial inhibitors. CD4+ T-cells were incubated for 24 h in the presence of medium containing IL-2 (10 U/ml), subsequently treated with SOD (2000 U/ml in the presence of 100 nM of digitonin), catalase (3000 U/ml), antimycin A (50 μM), or valinomycin (50 μM) for 1 h, after which TSA was added to the culture medium at growing concentrations. After 20 h, cells were harvested, and cell survival was determined by PI staining of nuclei. Shown are the mean percentage of survival; bars, SD. All data are representative of two independent experiments. A typical blot is shown.