Figure 3
COX deficiency leads to the accumulation of pro-apoptotic ceramides. (a) Total ceramide content of COX-deficient cells and their respective controls was analyzed by mass spectrometry in total cell homogenates. HeLa cells were pretreated with KCN (1 mM) for 48 h or left untreated. (b) Total ceramide synthase activity in COX-deficient cells and their respective controls was measured by the incorporation of d17 : 0 dihydrosphingosine into the sphingoid backbone of ceramides (d17 : 1 ceramides). Incorporation of d17 : 0 dihydrosphingosine (2 μM) into ceramides was assessed after 4 h by mass spectrometry of total cell homogenates. (c) COX-deficient cells were incubated in the presence of the ceramide synthase inhibitor FB1 (HeLa 20 μM, 143BΔCOX 30 μM and COX10−/− 50 μM) and HeLa cells were incubated additionally with KCN (1 mM) for 24 h. Cell death upon treatment with H2O2 was measured after 20 h by trypan-blue exclusion. Error bars in panel a represent the mean±S.D. of two analytical replicates of three biological replicates (HeLa cells and 143B cybrids) and mean±S.D. of two analytical replicates of two biological replicates (COX10), respectively. Error bars in panel b represent mean±S.D. of two analytical replicates of four biological (143B cybrids) and two biological (COX10) replicates, respectively. Error bars in panel c represent mean±S.D. (n=3), two-tailed unpaired t-test. *P<0.05, **P<0.01, ***P<0.001
