Journal: Journal of Lipid Research

Article Title: Sustained activation of sphingomyelin synthase by 2-hydroxyoleic acid induces sphingolipidosis in tumor cells ¹ [S]

doi: 10.1194/jlr.M036749

Figure Lengend Snippet: Effect on ceramide mass of the SMS inhibition by D609 in U118 cells. Inhibition of SMS by D609 in the presence of 2OHOA induced a minor accumulation of ceramides. The values represent the mean ± SEM (n = 3) and the asterisks (*) indicate a significant effect of treatment compared with controls: * P < 0.05. In this case, and indicates a significant effect of the inhibition compared with the treatment ( P < 0.05).

Article Snippet: U118 cells were incubated with D609 for 16 h (200 μM) and 2OHOA (200 μM) was added 1 h after the addition of D609 (Tocris Bioscience, UK).

Techniques: Inhibition

Journal: The Journal of Experimental Medicine

Article Title: Overexpression of Acid Ceramidase Protects from Tumor Necrosis Factor–Induced Cell Death

doi:

Figure Lengend Snippet: Pharmacological inhibition of AC diminishes protection from TNF-induced cell death, whereas inhibition of A-SMase enhances protection. (A) Parental and transfected L929 cells were incubated with or without 100 μM NOE in the presence of 100 ng/ml hTNF for 48 h. (B) Clones AC23 and AC52 were treated with 100 ng/ml hTNF with or without 100 μM NOE for 40 h before intracellular ceramide was determined. (C) Parental L929 cells were treated with or without 25 μM desipramine or 25 μg/ml D609 in the presence of 100 ng/ml hTNF for 48 h. Separately, L929 cells were incubated with 100 ng/ml hTNF for 48 h with or without addition of 50 μM fumonisin B1. Cell viability is expressed relative to untreated controls. Ceramide levels are expressed relative to untreated cells, in which basal ceramide was between 100 and 120 pmol per 10 7 cells. Values shown represent the means from three or six parallel determinations (ceramide levels or cell viability), and error bars indicate the respective SDs. * P < 0.002 versus cells not treated with NOE (determined by Student's t test). One out of three independent experiments with similar results is shown.

Article Snippet: D609 was purchased from MoBiTec and fumonisin B1 from Alexis Biochemicals.

Techniques: Inhibition, Transfection, Incubation, Clone Assay

Journal: Scientific Reports

Article Title: A non-toxic equinatoxin-II reveals the dynamics and distribution of sphingomyelin in the cytosolic leaflet of the plasma membrane

doi: 10.1038/s41598-024-67803-2

Figure Lengend Snippet: NT-EqtII exhibits a specific affinity for sphingomyelin located in the cytosolic leaflet of the living iMEF cell PM. ( a ) Schematic representation of NT-Eqt II observation in the cytosolic leaflet of the cell PM. ( b ) Fluorescent images of NT-EqtII-HaloTag7 labeled with SF650B, mEos4b-STING, and mEos4b-Rab6a in iMEFs. The images were acquired by oblique illumination (upper) and TIRF microscopy (TIRFM) (lower). Single fluorescent spots of NT-EqtII are indicated by yellow arrowheads. Scale bars, 2 μm. ( c ) Fluorescent images of NT-EqtII-HaloTag7 labeled with SF650B in various cellular contexts, encompassing wild-type (WT) iMEFs, SMS1 and 2-DKO iMEFs, iMEFs expressing WT bacterial SMase or its catalytically inactive mutant harboring Ras farnesylation sequence by which is anchored to the cytosolic leaflet of the PM, along with iMEFs subjected to D609 treatment. The images were acquired by oblique illumination (left) and TIRFM (right). Quantification of NT-EqtII-HaloTag7 expressed in cells was performed by evaluating fluorescence intensity via oblique illumination. The content of the SM probe in the cytosolic leaflet of the cell PM was quantified by measuring the numbers of single spots (yellow arrowheads) by TIRFM. Scale bars, 2 μm. ( d ) The numbers of localizations of NT-EqtII-HaloTag7 labeled with SF650B bound to the cytosolic leaflets of cell PMs, were determined by TIRFM at 4 ms/frame for 2000 frames and subsequently normalized by the expressed amounts (fluorescence intensities in the cytoplasm minus those in the background) measured by oblique illumination. The value in the vertical axis shows the difference between the numbers of localizations of NT-EqtII-Halo7-SF650B in cells expressing NT-EqtII and those in cells that do not express NT-EqtII but were incubated with SF650B. The presented error bars symbolize standard errors. *** and n.s. indicate significant (p < 0.001) and not significant differences (p > 0.05), respectively, using Welch’s T -test.

Article Snippet: D609 (CS-0078) was purchased from CEM.

Techniques: Labeling, Microscopy, Expressing, Mutagenesis, Sequencing, Fluorescence, Incubation

Journal: The Journal of Biological Chemistry

Article Title: Multiple activities of sphingomyelin synthase 2 generate saturated fatty acid– and/or monounsaturated fatty acid–containing diacylglycerol

doi: 10.1016/j.jbc.2024.107960

Figure Lengend Snippet: Effects of D609, a PC-PLC/SMS inhibitor, on PC-PLC, PE-PLC, SMS, and CPES activities of SMS2. A , DG-generating activities of SMS2, which was purified in the presence of detergents, were analyzed in the presence of various concentrations of D609 (0, 0.25, 0.5, and 1 mM). Purified WT SMS2-TS (approximately 500 ng) was incubated in the presence of 16:0/18:1-PC alone, 16:0/18:1-PC and d18:1/18:0-Cer, 16:0/18:1-PE alone, or 16:0/18:1-PE and d18:1/18:0-Cer–containing detergent mixed micelles for 2 h at 37 °C. Produced 16:0/18:1-DG was quantified using LC–MS/MS. Values are presented as percentages of the activity of SMS2 in the absence of D609 (set at 100%). Values are presented as the mean ± SD (n = 6, technical replicates). ∗∗∗, p < 0.005 ( versus control (without D609)). One-way ANOVA with Dunnett’s post hoc test was used. B , SM-producing activities of SMS2, which was purified in the presence of detergents, in the presence of 16:0/18:1-PC and d18:1/18:0-Cer–containing detergent mixed micelles were analyzed in the presence of various concentrations of D609 (0, 0.25, 0.5, and 1 mM). Values are presented as percentages of the SMS activity of SMS2 in the absence of D609 (set to 100%). The values are presented as the mean ± SD (n = 4, technical replicates). ∗, p < 0.05; ∗∗, p < 0.01 ( versus control (without D609)). One-way ANOVA with Dunnett’s post hoc test was used. C , CPE-producing activities of SMS2, which was purified in the presence of detergents, in the presence of 16:0/18:1-PE and d18:1/18:0-Cer–containing detergent mixed micelles were analyzed in the presence of various concentrations of D609 (0, 0.25, 0.5, and 1 mM). Values are presented as percentages of the CPES activity of SMS2 in the absence of D609 (set at 100%). Values are presented as the mean ± SD (n = 6, technical replicates). ∗∗, p < 0.01; ∗∗∗, p < 0.005 ( versus control (without D609)). One-way ANOVA with Dunnett’s post hoc test was used. Cer, ceramide; CPE, ceramide phosphoethanolamine; CPES, CPE synthase; DG, diacylglycerol; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PLC, phospholipase C; SM, sphingomyelin; SMS, sphingomyelin synthase; TS, Twin-Strep.

Article Snippet: Tricyclodecan-9-yl-xanthogenate (D609) was obtained from Chemscene.

Techniques: Purification, Incubation, Produced, Liquid Chromatography with Mass Spectroscopy, Activity Assay, Control

Journal: The Journal of Biological Chemistry

Article Title: Multiple activities of sphingomyelin synthase 2 generate saturated fatty acid– and/or monounsaturated fatty acid–containing diacylglycerol

doi: 10.1016/j.jbc.2024.107960

Figure Lengend Snippet: Comparison of SMS2 (our findings and previous reports), SMS1, and SMSr

Article Snippet: Tricyclodecan-9-yl-xanthogenate (D609) was obtained from Chemscene.

Techniques: Comparison, Membrane, Expressing, Activity Assay, In Vitro, Inhibition, Mutagenesis

Journal: The Journal of Biological Chemistry

Article Title: Multiple activities of sphingomyelin synthase 2 generate saturated fatty acid– and/or monounsaturated fatty acid–containing diacylglycerol

doi: 10.1016/j.jbc.2024.107960

Figure Lengend Snippet: Comparison of bacterial, plant, and mammalian PLCs

Article Snippet: Tricyclodecan-9-yl-xanthogenate (D609) was obtained from Chemscene.

Techniques: Comparison, Activity Assay, Membrane, Inhibition