Patterns of intracellular ceramide species induced by TNF-α or exogenous ceramides in lung endothelial cells. (a) Effect of TNF-α (20 ng/ml with cycloheximide, 16 h) or extracellular C8:0 ceramide (10 μM, 16 h) treatment on endogenous long-chain (C14:0 - C18:0) and very long–chain ceramides (C20–24). (b) Inhibitory effect of SPTLC1 and ASMase siRNAs on long-chain and very long–chain ceramides induced by TNF-α or C8:0 ceramide. Note the robust inhibition of very-long-chain ceramides induction by SPTLC1 siRNA (n = 2). (c) Pie charts of relative expression profiles of individual ceramide species comprising total ceramides measured by mass spectrometry. Ceramide species expression profiles at baseline and in response to C8:0 and TNF-α treatment. Depicted in the lower pie charts are the effects of SPTLC1 siRNA treatment on ceramide species induced by the two stimuli. (d) Changes in the relative abundance of C16:0 (% of total ceramides) induced by C8:0 and TNF-α treatment and the effect of SPTLC1 siRNA. The control values include untreated control, vehicle-treated control and scramble siRNA (mean ± SEM; *P < 0.05 versus controls, #P < 0.05 versus C8:0 and TNF-α treatment, respectively, Student's t test).