Figure 4.

DPE-mediated activation of neutral sphingomyelinase (SMase) is required for NOX activation-induced increase in ceramide and subsequent KC apoptosis. Human KC pre-treated with or without NOX (Apocynin [APO], 100 μM), ROS generation (N-Acetylcysteine [NAC], 1 mM), or GW4869 (neutral SMase inhibitor, 10 μM) for 30 mins were incubated with DPE (100 μg/mL) for 24 hrs. Activities of acidic and neutral SMases (A,B), and contents of ceramide and sphingosine-1-phosphate (D,E) were assessed by LC-ESI-MS/MS. Cell viability or cytotoxicity was measured by either WST-1 assay (C). All values are mean ± SD (n = 3). Statistical significance was calculated using the unpaired Student’s t-test, and significance was defined as p < 0.01 vs. vehicle control (or untreated control).