Figure 6.

Acid ceramidase (AC) inhibitor LCL385 blocks radiation-induced AC upregulation. (a) Dose-dependent inhibitory effect of LCL385 on AC in vitro was performed through a cell-free assay using PPC-1 cell homogenates as the AC source and [³H-N] labeled C₆ ceramide as the substrate, performed at pH 4.5. Data are plotted as percentage of AC inhibition compared to control cell lysate. (b) Cytotoxicity of LCL385 on PPC-1 cells was evaluated by MTS assay 24 hours following treatment at the indicated doses. Results represent percentage of cell death compared to cells with no treatment. (c) PPC-1 cells were treated with LCL385 (2.5 µmol/l). At the indicated time points sphingosine levels were measured in triplicate using mass spectrometry and plotted as the percent change compared to cells with no LCL385 treatment. (d) PPC-1 cells were treated with LCL385 (2.5 µmol/l) and protein lysates were isolated at the indicated time points and processed by western blotting for AC protein expression. (e) PPC-1 cells were treated with LCL385 (2.5 µmol/l) 8 hours prior to radiation as indicated. Protein lysates were collected at 8 or 24 hours after radiation administration and processed by western blotting to determine AC protein levels.