Inhibitors of acid sphingomyelinase suppress the RGDfV-induced increase in ceramide. (A) HBMECs (10⁶ cells/10-cm plate) were allowed to spread on dishes coated with vitronectin and blocked with 1% BSA. Cells were labeled for 24 hours with [³H]palmitic acid, washed, equilibrated without isotope for 2 hours, washed again, and incubated with RGDfV (G; ▪; 25 μg/mL), RADfV (A; ▧), or vehicle control (-; □) for 24 hours. Lipids were extracted, and [³H]ceramide and [³H]sphingomyelin of each sample were determined by TLC as described in “Materials and methods.” ^*P < .001 ([³H]ceramide) and ^**P = .029 ([³H]sphingomyelin) compared with the corresponding RADfV or vehicle control, by unpaired t test; n = 3, representative experiment of 5 experiments, each performed in triplicate. (B) HBMECs were seeded on vitronectin, labeled with [³H]palmitic acid, and exposed to RGDfV (G, ▪), RADfV (A, ▧), or vehicle control (-, □), as described in panel A. The 2 RGDfV-treated triplicate samples were also incubated with imipramine (I) or desipramine (D) (20 μM; ▦) starting 2 hours prior to addition of RGDfV. [³H]ceramide was assessed after overnight incubation as in panel A. ^*P = .001 and ^**P = .001 compared with RGDfV. The dashed line depicts the level of baseline cellular ceramide content in HBMECs treated with the RADfV control peptide.