Fig. 3.

JB inhibits CerS activity. A: CerS activity was determined in cell lysates overexpressing different CerSs. Lysates (CerS1, 25 μg; CerS2, 40 μg; CerS4, 30 μg; CerS5, 1 μg; CerS6, 5 μg) were preincubated for 5 min with JB (5 μM) or ethanol as a control; the reaction was started by adding NBD-dhSo (15 μM)/BSA (20 μM)/acyl-CoA (50 μM) (CerS1, C18 acyl-CoA; CerS2, C22 acyl-CoA; CerS4, C18 or C20 acyl-CoA; CerS5, C16 acyl-CoA; CerS6, acyl-CoA) to the samples. The reaction was carried out during different times (CerS1, 20 min; CerS2, 10 min; CerS4, 20 min; CerS5, 5 min; CerS6, 10 min). Results are the mean ± SD for two experiments performed in duplicate and are expressed as the percent of the activity compared with the control. *P < 0.05, **P < 0.005, ***P < 0.0005. B: Cell lysates overexpressing CerS5 were incubated for 20 min with DhSo or JB at 15 μM and with C16-[1-¹⁴C]acyl-CoA (0.01 uCi)/C16 acyl-CoA(46 μM) and BSA (20 μM). Results are representative of a typical experiment in duplicate (n = 2). C: HGC-27 cells were treated with 1 μM JB for 4, 8, and 24 h. Lipids were extracted and analyzed by UPLC/TOF. Data are given as picomolar equivalents to C12:0 Cer and are the mean of one experiment in triplicate, which is representative of three experiments that gave similar results.