Figure 6.

Inhibition of SPHK1 alters multiple survival pathways in leukemic NKL cells. (A) NKL cells were treated for the indicated time with either DMSO or SKI-178 (5 μM) and then harvested for protein. Equal amounts of protein lysate were analyzed by western blotting with antibodies against P-JNK, JNK, P-c-JUN, P-p38 MAPK, p38 MAPK, P-ERK1/2 and ERK1/2. Equal loading confirmed by total JNK, p38 and ERK1/2 levels and β-Actin. (B) Dose-dependent treatment of NKL or RNK-16 cells with SKI-178 for 24 hours. Equal amounts of protein lysate were analyzed by protein gel blotting with antibodies against P-JNK, JNK, P-p38 MAPK, p38 MAPK, P-ERK1/2 and ERK1/2. Equal loading of protein was confirmed by probing for β-actin. C and D) NKL cells were pretreated for 1 hour with a JNK inhibitor, SP600125 (SP) at 10 μM, a CDK1 inhibitor, Ro3306 at 10 μM, or a second JNK inhibitor, JNK inhibitor XVI at 10 μM. The cells were then incubated with SKI-178 (5 μM) or DMSO for 48h and cell viability assessed by MTS assay (C) or Annexin V/7-AAD apoptosis by flow cytometry (D E) NKL cells were pretreated with SP (10 μM), Ro3306 (10 μM) or JNK inhibitor XVI (10 μM) for 1 hour and then incubated with SKI-178 (5 μM) or DMSO for 24 hours. Cells were harvested for protein lysate and equal amounts of protein were analyzed by western blotting with antibodies against P-Bcl-2 (Ser70) and Bcl-2. Equal loading was confirmed by β-actin. *P < 0.05 indicates significant differences as shown (Student's t test).