Fig. 5.

The role of EGFRvIII in growth and SphK1 regulation in GBM-derived neurosphere cells. a GBM9 and GBM44 were plated at 50,000 cells per well and cultured for the indicated time. Cells per well were counted. b GBM9 cells were treated with DMSO as a vehicle control or the indicated concentration of AG1478 for 2 days and cell proliferation was measured by MTT assay. The insets in panels b and f show the effects of AG1478 and gefitinib on EGFRvIII tyrosine phosphorylation as determined by phospho-EGFR-specific Western blot analysis. c GBM9 cells were treated with 1 μM AG1478 for 2 days and SphK1 activity was measured, or real time PCR was performed to measure SphK1 mRNA level. d and e GBM9 cells were grown for 2 days in the presence of vehicle or the indicated concentration of SphK inhibitor and SphK activity was assayed (d) or cell proliferation was measured by MTT assay (e) in parallel wells. f GBM9 cells were grown for 3 days in the presence of the indicated concentration of gefitinib or DMSO as a vehicle control and cell proliferation was measured by MTT assay. Results for all panels are means ± s.d. of triplicate samples. The * indicate statistically significant difference by Student’s T test, p < 0.05