Figure 6
Figure 6. Ceramide causes ASK1-regulated p38 MAPK and JNK activation, and p38 MAPK- and JNK-mediated apoptosis. (A) Jurkat T cells were treated with C2-ceramide for different time periods as indicated. The expression of phospho-p38 MAPK (pp38 MAPK) at threonine180/tyrosine182, p38 MAPK, phospho-JNK (pJNK) at threoine183/tyrosine185, JNK, and Txnip were determined using Western blot analysis. The relative ratios of pp38 MAPK to p38 MAPK and pJNK to JNK are shown. The expression of β-actin was an internal control. (B) Vector control– and ASK1 siRNA-transfected Jurkat T cells were treated with 20 μM C2-ceramide for 6 hours, and the expression of pp38 MAPK and pJNK were determined by flow cytometric analysis. The percentages of positive cells are shown as means plus or minus SD of triplicate cultures (**P < .01 as compared with the vector control group). (C) Cells were pretreated with SB203580 (25 μM) or SP600125 (25 μM) for 1 hour, followed by C2-ceramide treatment for 24 hours. Apoptotic cells were detected using PI staining followed by flow cytometric analysis. The percentages of apoptotic cells are shown as means plus or minus SD of triplicate cultures (*P < .05 as compared with the groups without inhibitor treatment). (D) Meanwhile, activation of caspase-9 and caspase-3 at 24 hours as determined by caspase activity assay kit are shown (means ± SD of triplicate cultures). The mitochondrial transmembrane potential (ΔΨm) reduction in cells with C2-ceramide stimulation for 12 hours was detected by rhodamine 123 followed by flow cytometric analysis, and are shown as means plus or minus SD of triplicate cultures (*P < .05; ***P < .001 as compared with the groups without inhibitor treatment). (E) The p38 MAPK and JNK protein expression in vector control– and p38 MAPK or JNK siRNA-transfected Jurkat T cells were detected by Western blotting (top). The relative ratios of p38 MAPK to EGFP and JNK to EGFP are shown. The expression of α-tubulin was used as an internal control. Cells were treated with 20 μM C2-ceramide for 24 hours, and cell apoptosis was measured by annexin V staining. The percentages of apoptotic cells are shown as means plus or minus SD of triplicate cultures (*P < .05 as compared with the vector control group).

Ceramide causes ASK1-regulated p38 MAPK and JNK activation, and p38 MAPK- and JNK-mediated apoptosis. (A) Jurkat T cells were treated with C2-ceramide for different time periods as indicated. The expression of phospho-p38 MAPK (pp38 MAPK) at threonine180/tyrosine182, p38 MAPK, phospho-JNK (pJNK) at threoine183/tyrosine185, JNK, and Txnip were determined using Western blot analysis. The relative ratios of pp38 MAPK to p38 MAPK and pJNK to JNK are shown. The expression of β-actin was an internal control. (B) Vector control– and ASK1 siRNA-transfected Jurkat T cells were treated with 20 μM C2-ceramide for 6 hours, and the expression of pp38 MAPK and pJNK were determined by flow cytometric analysis. The percentages of positive cells are shown as means plus or minus SD of triplicate cultures (**P < .01 as compared with the vector control group). (C) Cells were pretreated with SB203580 (25 μM) or SP600125 (25 μM) for 1 hour, followed by C2-ceramide treatment for 24 hours. Apoptotic cells were detected using PI staining followed by flow cytometric analysis. The percentages of apoptotic cells are shown as means plus or minus SD of triplicate cultures (*P < .05 as compared with the groups without inhibitor treatment). (D) Meanwhile, activation of caspase-9 and caspase-3 at 24 hours as determined by caspase activity assay kit are shown (means ± SD of triplicate cultures). The mitochondrial transmembrane potential (ΔΨm) reduction in cells with C2-ceramide stimulation for 12 hours was detected by rhodamine 123 followed by flow cytometric analysis, and are shown as means plus or minus SD of triplicate cultures (*P < .05; ***P < .001 as compared with the groups without inhibitor treatment). (E) The p38 MAPK and JNK protein expression in vector control– and p38 MAPK or JNK siRNA-transfected Jurkat T cells were detected by Western blotting (top). The relative ratios of p38 MAPK to EGFP and JNK to EGFP are shown. The expression of α-tubulin was used as an internal control. Cells were treated with 20 μM C2-ceramide for 24 hours, and cell apoptosis was measured by annexin V staining. The percentages of apoptotic cells are shown as means plus or minus SD of triplicate cultures (*P < .05 as compared with the vector control group).

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