Fig. 2.

Direct and specific inhibition of ASM by ARC39 under cell culture conditions. A: L929 cells were treated for 2 h with ARC39 as indicated, and the activity of ASM, NSM, and NC was determined. L929 cells were treated for 2 h with ARC39, amitriptyline (Ami) or desipramine (Desi) as indicated, and AC activity was subsequently determined in B, and AC protein was analyzed by Western blotting in C. D: L929 cells were treated with 20 μM of ARC39 as indicated: fold change of Smpd1 mRNA relative to Hprt1 (left), ASM protein level (representative Western blot) (right). E: ASM activity in L929 cells after treatment with ARC39 as indicated. P < 0.01 for treatment time as a source of variation in ASM activity. F: L929 cells were preincubated for 24 h with 25 μM of leupeptin or vehicle then treated for 4 h, as indicated, and ASM activity was subsequently determined. Enzyme activity is normalized to the protein concentration, and control values are normalized to their mean. ASM activity shown in this figure is determined with the conventional assay without addition of zinc and is attributed to L-ASM. Data are represented as mean ± SD, n = 3–5 experiments. One-way ANOVA was used in B and F and two-way ANOVA in D and E; both were followed by Bonferroni correction (except for ASM activity). *P < 0.05, ***P < 0.001, ****P < 0.0001.