Fig 1: Functional validation of ASM inhibition by ARC39: ARC39 inhibits platelet- and ASM-promoted adhesion of tumor cells. B16F10 melanoma cells (4 × 104) were incubated for 5 min at 37°C in 100 μl total volume with 2 × 107 WT Asm-deficient (Asm−/−) platelets (Plts) or rhASM (0.125 μg in 50 μl) in the presence or absence of 1 μM of ARC39, which was added directly upon coincubation without previous pretreatment. After addition of 300 μl of B16F10 MEM complete culture medium (also containing 1 μM of ARC39 where indicated), tumor cells were incubated for 3 min on fibronectin-coated coverslips, washed, fixed, and stained with DAPI. Adherent tumor cells were then counted. Data are represented as mean ± SD, n = 3–4 experiments. One-way ANOVA was used followed by Tukey’s correction. ****P < 0.0001.
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.
Fig 3: Direct and selective inhibition of L-ASM and S-ASM by ARC39 in micellar assays. A: Chemical structure of ARC39. B: L929 whole-cell lysates were treated with ARC39 as indicated, and the activity of L-ASM (no ZnCl2 was added to the assay buffer), NSM, AC, and NC was determined. C: Mouse serum was treated with ARC39 as indicated and the activity of S-ASM (+100 μM ZnCl2 in the assay buffer) and AC was determined. D: L929-derived supernatant medium (L-sup) and FCS were treated as indicated with or without addition of ZnCl2 to the assay buffer and the activity of S-ASM was determined. E, F: L929 cell lysates or mouse serum, respectively, were treated with PBS control or 80 μM of amitriptyline and the activity of ASM (L-ASM in cell lysates and S-ASM in serum), NSM, AC, and NC was determined. Enzyme activity is normalized to the protein concentration and control values are normalized to their mean except in D where they are expressed as absolute values. Data are represented as mean ± SD, n = 3 experiments.
Fig 4: Inhibition of L-ASM in living cells by ARC39. A, B, D: After the indicated treatments for the indicated durations, cells were incubated with the FRET probe at a final concentration of 1 μM for 30 min (L929) or 1 h (HepG2) and were then washed, briefly trypsinized, and analyzed by flow cytometry. A: Comparison of the dose response in L929 cells treated with ARC39 for 2 h or 24 h. P < 0.0001 for time as a source of variation in ASM activity. B: Effect of the treatment duration on ASM inhibition by ARC39. L929 cells were treated with 20 μM of ARC39 for the indicated times, and then ASM activity was determined with the FRET probe. C: Mass spectrometric analysis of the ratio of total sphingomyelins/total ceramides (SMs/Cers) in whole-cell lysates from L929 cells that had been treated for 24 h with ARC39 as indicated. D: Comparison of the dose-response in HepG2 cells treated with ARC39 for 2 h or 24 h. P < 0.05 for time as a source of variation in ASM activity. E: Representative histogram overlay for the MFI of the FRET probe in both green (520 nm) and red (700 nm) channels from HepG2 cells treated as indicated for 2 h. F: L929 cells were incubated for 24 h with 1 μM of BODIPY FL sphingomyelin (SM) together with PBS as control, 10 μM of ARC39 or 10 μM of amitriptyline (Ami) as a positive control. Then, the cells were incubated for 30 min at 37°C with 25 nM of LysoTracker Red DND-99. Fresh medium was added, and then the cells were analyzed by confocal microscopy (magnification 100×). Control values are normalized to their mean. Data are represented as mean ± SD, n = 3–4 experiments. One-way ANOVA was used in B and C followed by Bonferroni correction, and two-way ANOVA was used in A and D. *P < 0.05, **P < 0.01, ***P < 0.001, #P < 0.0001.
Fig 5: ASM inhibition by ARC39 in vivo. ARC39 was applied intraperitoneally every 12 h for 96 h at a dose of 0.16 mM 10 μl/g and sphingomyelin content was determined by MS 12 h after the last injection. Data are represented as mean ± SD, n = 4 mice per group. Unpaired Student’s t-test was used. *P < 0.05 **P < 0.01.
Supplier Page from R&D Systems, a Bio-Techne Brand for Recombinant Human SMPD1 Protein, CF