Etoposide induces ceramide generation, ceramide- and p38 MAPK/JNK-dependent cell apoptosis, and Txnip up-regulation. (A) Jurkat T cells were treated with 25 μM etoposide for different time periods as indicated, and the generation of ceramide was determined by flow cytometric analysis. After fixation with 1% formaldehyde in PBS, cells were incubated with ceramide antibodies, followed by FITC-labeled secondary antibodies. Representative histograms (top) and the percentages of positive cells (bottom) are shown (means ± SD of triplicate cultures; *P < .05; **P < .01). (B) Jurkat T cells were pretreated with fumonisin B1, myriocin, chlorpromazine, desprimine, SB203580, or SP600125 for 1 hour, followed by etoposide treatment for 24 hours. Apoptotic cells were detected using PI staining followed by flow cytometric analysis. The percentages of apoptotic cells are shown (means ± SD of triplicate cultures; ***P < .001). (C) The Txnip and thioredoxin protein expression after 25 μM etoposide treatment for different time periods was detected using Western blot analysis. The expression of β-actin was used as an internal control. The relative ratio of Txnip–β-actin and thioredoxin–β-actin are shown.