Fig. 6.

Changes in the metabolism of sphingolipids and eicosanoids in BMMCL with SPTLC1 or 5-LO knockdowns (KDs). A: Lysates from BMMCL transduced with empty vector (control), SPTLC1 shRNA (SPTLC1 KD), and 5-LO shRNA (5-LO KD) were assessed by immunoblotting with the indicated antibodies. B–E: Quantification of data as in A, normalized to expression in controls and HPRT load. Controls (n = 9), SPTLC1 KD (n = 8), and 5-LO KD (n = 8). F–L: LC-ESI-MS/MS analysis of sphingolipids in Ag-activated transduced BMMCL: control (n = 8), SPTLC1 KD (n = 8), and 5-LO KD (n = 9). F: The sum of total sphingosines, C18:1, C18:0, C20:1, and C20:0, is calculated. G–J, The values of distinct sphingosines C18:1 and C20:1 and sphinganines C18:0 and C20:0 are shown. K: The sum of total ceramide fatty acid chain molecular species, derived from C18:1 sphingosine. L: Non-2-hydroxylated ceramide molecular species derived from C18:1 sphingosine. M–P: UPLC MS/MS analysis of eicosanoids from supernatants of Ag-activated BMMCL: controls (n = 8; except TXB₂ n = 7), SPTLC1 KD (n = 8; except TXB₂ n = 7), and 5-LO KD (n = 7; except TXB₂ n = 6). Quantitative data are mean ± SEM, calculated from n, which show numbers of biological replicates of independently transduced cells. P values were determined by one-way ANOVA with Bonferroni post hoc test except for LTB₄, 6t-LTB₄, and LTB₅ data, which were compared by nonparametric Kruskal-Wallis test with Dunn's post hoc test.