Figure 7.

Essential role of nSMase1 in stress-induced ceramide generation in human Jurkat T cells in a loss-of-function study. (a) Knockdown of SMPD2 expression in human Jurkat T cells. (b) Detection of phosphorylated-nSMase1 by western blotting. The cells were transfected with control or SMPD2 RNAi and cultured for 24 h. The transfected cells were stressed using heat shock at 43 °C for 30 min, UV-irradiated at 5 mJ/cm² for 30 min, 1 mM H₂O₂ treatment for 30 min, and stimulation with 50 ng/ml anti-Fas antibody for 3 h. Cell lysates were harvested and analyzed by western blotting with anti-phospho-nSMase1, anti-nSMase1, anti-phospho-JNK, anti-JNK, anti-phospho-c-jun, or anti-actin antibodies. Molecular weight markers are shown in kDa on the right. (c) nSMase1 activation by SMPD2 knockdown. The cell lines were heat-shocked at 43 °C for 0, 15, 30, or 60 min, irradiated at 5 mJ/cm² UV for 0, 15, 30, or 60 min, treated with 1 mM H₂O₂ for 0, 15, 30, or 60 min, and stimulated with 50 ng/ml of anti-Fas antibody for 0, 15, 30, or 60 min. The cells were then incubated at 37 °C for up to 5 h. (d) Ceramide generation after SMPD2 knockdown. (e and f) Effect of SMPD2 knockdown on the induction of apoptosis in human Jurkat T cells. Each value represents the mean of three independent experiments, and the error bars represent the S.D.s. *P<0.01 versus the control