Effects of a constitutively active PI-3 kinase on cell growth, ceramide content, and activities in GCS, SMS, and acid and neutral SMases in COS-7 cells. The expression vector for constitutively activated PI-3 kinase (PI-3K active) was transiently transfected to the COS-7 cells by electroporation method as described in “Materials and methods.” (A) PI-3 kinase-dependent protein expression of phosphorylated Akt (Serine 473) was determined by Western blot analysis, using antibody for Akt and phospho-Akt (Serine 473) antibodies. (B) Transfected COS-7 cells were seeded in the 24-well culture dish at an initial concentration of 2 × 10⁴ cells/wells and cultured for 48 hours. Viable cell numbers were counted by a trypan blue dye exclusion method at the indicated times (0, 24, and 48 hours). (C) Ceramide content in COS-7 cells 24 hours after transfection was measured by DGK assay as described in “Materials and methods.” Ceramide content in the mock cells was shown as the control (5.8 ± 1.2 pmol/nmol phosphate). The activities of GCS (D), SMS (E), and acid (F) and neutral (G) SMases in COS-7 cells after 24 hours of transfection were measured by using C6-NBD-ceramide and C6-NBD-SM as described in “Materials and methods.” The activities of each enzyme in the mock cells were shown to be 1564 ± 133, 359 ± 28, 664 ± 28, and 30 ± 0.84 pmol/mg protein/hour for GCS, SMS, and acid and neutral SMases, respectively. In these experiments 75% ± 9% was a transfection efficiency as judged by expression of transfected β-galactosidase. The results were obtained from 3 different experiments. The bars indicate 1 SD. The significance of difference was determined by ANOVA test. ^*P < .01.