Effect of C6-ceramide, tamoxifen, and ethylenedioxy-P4 on 2780AD cell viability. (A) Viability. Cells in 96-well plates were exposed to the agents shown (C6-ceramide, 10 μg/ml; tamoxifen 5 μM) for 24 hr, after which viability was measured by MTS assay. (B) Effect of tamoxifen and GCS inhibitor ethylenedioxy-P4 on C6-GC synthesis from C6-ceramide. 2780AD cells in 6-well plates were exposed to the agents indicated (tamoxifen, 5 μM) for 24 hr in medium containing [14C]C6-ceramide (1.0 μg/ml medium). [14C]C6-GC synthesis was measured by TLC of total lipid extracts. (C) Effect of high-dose ethylenedioxy-P4 on C6-ceramide cytotoxicity in 2780AD cells. Cells were exposed to 10 μg/ml C6-ceramide and the concentrations of ethylenedioxy-P4 indicated, for 24 hr. Viability was determined by MTS. Results are the mean ± S.D. of six replications for viability determinations. Repeated experiments yielded similar results. C6-GC synthesis results are the mean ± S.D., n = 3.