Effect of ceramide synthesis inhibition on TNF-α–induced effector caspase activation in pulmonary endothelial cells. Primary mouse lung endothelial cells were pretreated with (a) SPTLC1 siRNA (50 nM, 6 d) or (b) ASMase siRNA (50 nM, 72 h) before TNF-α treatment (20 ng/ml, with cycloheximide). Caspase-3/7 activity was normalized by protein concentration; Veh (vehicle siPortamine). (c, d) Effect of siRNA inhibitory treatments on intracellular ceramides (normalized by lipid phosphorus). Effect of Scramble siRNA (Scr siRNA, 50 nM, 6 d) or SPTLC1 siRNA (50 nM, 6 d) on TNF-α-induced ceramides (c) compared with the effect of ASMase siRNA (50 nM, 3 d) (d). SPTLC1, but not ASMase inhibition significantly reduced ceramide up-regulation in response to TNF-α (20 ng/ml, with cycloheximide, 16 h); (mean ± SD, *P < 0.05 versus control untreated cells; #P < 0.05 versus control TNF-α–treated cells).