Figure 5.

The ISO- and 8CPT-2Me-cAMP–induced action on cell motility is dependent on PTEN expression in glioma cells. (A) Detection of PTEN by Western blot analysis. Cell extracts were prepared from human U87MG cells, 1321N1 astrocytoma cells, GNS-3314 cells, CGNH-89 cells, and rat C6 glioma cells. (B) CGNH-89 cells were serum-starved and subjected to migration assay. The cells were stimulated with or without 100 nM LPA in the presence or absence of 1 μM ISO, 30 μM 8CPT-2Me-cAMP (8CPT), and 100 nM S1P. (C) C6 cells were serum-starved and stimulated with or without 100 nM LPA in the presence or absence of 1 μM ISO and 30 μM 8CPT-2Me-cAMP (8CPT). (D–G) U87MG cells were serum-starved and stimulated with the indicated agents to measure migration activity (D), Rac1 activity by the pulldown assay (E), Akt phosphorylation activity in (F), and Rap1 activity by the pulldown assay (G). The concentrations of test agents and other experimental conditions were the same as those of B for D, Figure 1C for E, Figure 4C for F, and Figure 2C for G. (H) U87MG cells were transfected with pEGFP-C1 containing PTEN or plasmid alone (GFP) according to Nucleofector Kit T, cultured in 0.1% BSA/DMEM, and then incubated to measure the migratory response to a vehicle and 100 nM LPA in the presence or absence of 1 μM ISO. (I) 1321N1 cells were serum-starved and treated with or without 100 nM LPA in the presence or absence of 1 μM ISO, 30 μM 8CPT-2Me-cAMP (8CPT), and 100 nM S1P. The LPA effect was significantly lower than that in control cells (*) in B–D, H, and I.