Fig. 7. Cell-permeable ceramides or treatment with bacterial SMase induced death by a caspase-3-independent mechanism. (A) A20 cells were incubated for 16 hours with 0-120 µM C6-ceramide (open circles) or dihydro-C6-ceramide (closed circles). Cell viability was determined by the MTS assay and expressed as percentage of cell viability observed for non-treated cells. (B) Caspase-3 activity was determined after treatment of A20 cells for 4 hours either with C6-ceramide at the concentrations indicated, with 80 µM dihydro-C6-ceramide (DH), with 100 ng/ml FasL (F), with 100 ng/ml FasL and 80 µM C6-ceramide (F/C6) or remained untreated. Values are shown as a percentage of the caspase-3 activity in the presence of 100 ng/ml FasL alone. (C) Alternatively, DNA fragmentation was analyzed by electrophoresis in agarose gels after treatment with FasL or different concentration of ceramides for 4 hours. (D) A representative experiment is shown where A20 cells were treated with 100 µM C6- (black) or dihydro-C6 (gray) ceramide for 16 hours and cell survival was determined by flow-cytometric analysis following PI staining. (E) A20, Jurkat and Raji cells were either not treated (NT) or treated with 100 µM C6-ceramides (C6) or 100 ng/ml FasL (F) for 16 hours. Then cells were harvested, stained with PI and cell survival was determined by flow cytometric analysis. (F) Cells were treated with bacterial SMase at the concentrations indicated for 16 hours and cell viability was determined by the MTS assay. (G) In parallel, cells either not treated (gray) or treated with 0.5 U/ml bacterial SMase (black) were stained with PI, and cell survival was determined by flow cytometric analysis. The results shown are either the means or standard deviations of three independent experiments (A,B,E,F) or are representative of at least three independent experiments (C,D,G).