Fig. 3.

Amidase activity of different AML cell lines over RBM1-151. A: Cells were treated with SACLAC (15 μM) or vehicle (DMSO) for 1 h followed by 1 h incubation with RBM1-151 (20 μM). Cells were then processed for fluorescence analysis as detailed in the Materials and methods section. Data correspond to the mean ± SD of three experiments in triplicate. Asterisks indicate statistical difference at P ≤ 0.0001 (unpaired, two-tailed t-test vs. vehicle). B: AC activity. In the data of panel A, each SACLAC value was subtracted from the mean DMSO value. Asterisks indicate statistical difference at ∗P ≤ 0.001 and ∗∗P ≤ 0.0001 (one-way ANOVA, Tukey multiple comparisons test). C: MM-6 cells were treated with SACLAC (2.5 μM) or URB597 (50 μM) for the indicated times. The media were renewed, and hydrolysis of RBM1-151 (15 μM, 1 h) was determined as detailed in the Materials and methods section. The data (mean ± SD of one representative experiment with triplicates) were analyzed by the one-phase decay equation to afford the Y0 and plateau values. D: Estimated AC activity: 2.44 ± 0.44 μM/h/2 × 10⁴ cells (Y0—plateau in SACLAC treatment); estimated FAAH activity: 3.03 ± 0.62 μM/h/2 × 10⁴ cells (Y0—plateau in URB597 treatment) and estimated NAAA activity: 1.49 ± 0.63 μM/h/2 × 10⁴ cells (plateau in SACLAC treatment—FAAH activity) or 1.44 ± 0.65 μM/h/2 × 10⁴ cells (plateau in URB597 treatment—AC activity). AC, acid ceramidase; AML, acute myeloid leukemia; FAAH, fatty acid amide hydrolase; NAAA, N-acylethanolamine-hydrolyzing acid amidase; SACLAC, (2S,3R)2-chloro-N-(1,3-dihydroxyoctadecan-2-yl)acetamide.