Figure 6.

An essential role for MAPK8 and MAPK9 in nSMase1 activation and ceramide generation in human Jurkat T cells in a loss-of-function study. (a) Detection of phosphorylated-nSMase1 by western blotting after the RNAi-mediated knockdown of MAPK8 and/or MAPK9 in Jurkat T cells. Jurkat T cells were transfected with control, MAPK8, and/or MAPK9 RNAi and then heat-shocked at 43 °C for 30 min, UV-irradiated at 254 nm and 5 mJ/cm² for 30 min, treated with 1 mM H₂O₂ for 30 min, or stimulated with 50 ng/ml of anti-Fas antibody for 3 h. Cell lysates were harvested and analyzed by western blotting with anti-phospho-nSMase1, anti-nSMase1, anti-JNK, anti-phospho-c-jun, or anti-actin antibodies. Molecular weight markers are shown in kDa on the right. MAPK8 and MAPK9 are the human genes encoding JNK1 and JNK2, respectively. (b) nSMase activation. RNAi-transfected cell lines were stressed as in panel (a) and then incubated at 37 °C for up to 5 h. (c) Ceramide generation. (d) Effect of loss of function of MAPK8 and MAPK9 on the induction of apoptosis. Twenty-four hours after RNAi transfection, apoptosis was quantified in the MAPK8- and/or MAPK9-knockdown cells using DAPI staining after stress treatment as described in panel (a). (e) Effect of loss of function of MAPK8 and MAPK9 on apoptosis induction assessed using a caspase-3 assay. Each value represents the mean of three independent experiments, and the error bars represent the S.D.s. *P<0.01 versus control RNAi