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Figure Lengend Snippet: 2OHOA does not activate SMS (tissue homogenates). A, liver SMS activity measurement. Mouse liver homogenate was incubated with 2OHOA (200 μm) together with NBD-ceramide. NBD-SM, the product of SMS, was separated by TLC. B, quantification of SMS activity. C, HEK293 cell SMS activity measurement with the indicated dose of 2OHOA. D, HeLa cell SMS activity measurement with the indicated dose of 2OHOA. Values are mean ± S.D. (error bars), n = 5; *, p < 0.05.

Article Snippet: The homogenate was centrifuged at 2000 rpm for 10 min at 4 °C, and the cell supernatant (200 μg of protein) was mixed in assay buffer containing 100 m m Tris-HCl (pH 7.4), 50 m m KCl, 1 m m EDTA, C6-NBD-ceramide (4.3 μ m ) (Avanti Polar Lipids), and phosphatidylcholine (95 μ m ) (Sigma).

Techniques: Activity Assay, Incubation

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Figure Lengend Snippet: 2OHOA does not activate SMS (ex vivo). U118 cells (A) and A549 cells (B) were treated with different concentration of 2OHOA for 24 h, and cell mortality was monitored by an MTT assay. Cells were treated with 200 μm 2OHOA, and then NBD-ceramide was added to the cell culture medium and incubated for 30 min. SMS activity was measured in U118 cells (C), A549 cells (D), and macrophage cells (E). Values are mean ± S.D. (error bars), n = 3–5.

Article Snippet: The homogenate was centrifuged at 2000 rpm for 10 min at 4 °C, and the cell supernatant (200 μg of protein) was mixed in assay buffer containing 100 m m Tris-HCl (pH 7.4), 50 m m KCl, 1 m m EDTA, C6-NBD-ceramide (4.3 μ m ) (Avanti Polar Lipids), and phosphatidylcholine (95 μ m ) (Sigma).

Techniques: Ex Vivo, Concentration Assay, MTT Assay, Cell Culture, Incubation, Activity Assay

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Figure Lengend Snippet: 2OHOA does not activate purified SMS. SMS1-His tag and SMS2-His tag were purified from transfected insect cells (see “Experimental procedures”). A, SMS activity of SMS1-His tag and SMS2-His tag. SMS1-His tag and SMS2-His tag (1 μg) were incubated with 200 μm 2OHOA for 2 h, and then SMS activity was measured by adding NBD-ceramide. NBD-SM was the product. B, effect of 2OHOA on SMS1-His tag. C, effect of 2OHOA on SMS2-His tag. D and E, quantification of SMS activity by SMS1-His tag and SMS2-His tag. Values are mean ± S.D. (error bars), n = 3.

Article Snippet: The homogenate was centrifuged at 2000 rpm for 10 min at 4 °C, and the cell supernatant (200 μg of protein) was mixed in assay buffer containing 100 m m Tris-HCl (pH 7.4), 50 m m KCl, 1 m m EDTA, C6-NBD-ceramide (4.3 μ m ) (Avanti Polar Lipids), and phosphatidylcholine (95 μ m ) (Sigma).

Techniques: Purification, Transfection, Activity Assay, Incubation

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Figure Lengend Snippet: The in vivo effect of 2OHOA. A and B, effect on liver and small intestine (SI) total SMS activity (using NBD-ceramide (Cer) as substrate). C and D, effect on liver and SI total LPCAT activity (using oleoyl-CoA and NBD-lyso-PC as substrates) and quantification. Values are mean ± S.D. (error bars), n = 4–5. *, p < 0.001.

Article Snippet: The homogenate was centrifuged at 2000 rpm for 10 min at 4 °C, and the cell supernatant (200 μg of protein) was mixed in assay buffer containing 100 m m Tris-HCl (pH 7.4), 50 m m KCl, 1 m m EDTA, C6-NBD-ceramide (4.3 μ m ) (Avanti Polar Lipids), and phosphatidylcholine (95 μ m ) (Sigma).

Techniques: In Vivo, Activity Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Targeting sphingosine kinase 2 (SphK2) by ABC294640 inhibits colorectal cancer cell growth in vitro and in vivo

doi: 10.1186/s13046-015-0205-y

Figure Lengend Snippet: The effect of ABC294640 on SphK activity, S1P or ceramide content in CRC cells. The relative SphK activity ( a and b ), S1P content ( c ) or ceramide level ( d ) (vs. Control group) with indicated ABC294640 treatment were presented. The effect of S1P (5 μM), C6 ceramide (C6, 5 μM) or SKI-II (5 μM) on ABC294640 (1 μM)-induced HT-29 cell growth inhibition and cell death were tested by MTT assay ( e ) and LDH release assay ( f ), respectively. Mean values ± SD of three independent experiments were reported. Statistical analysis was performed comparing treatment groups with vehicle control group (“C”). *P < 0.05. # P < 0.05 vs. ABC294640 only group ( e and f )

Article Snippet: Cell permeable short-chain C6 ceramide was obtained from Avanti Polar Lipids, Inc. (Alabaster, AL).

Techniques: Activity Assay, Inhibition, MTT Assay, Lactate Dehydrogenase Assay

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Figure Lengend Snippet: FIGURE 2. Ceramide synthesis in CerS2 null mouse liver. The synthesis of C22- or C24-ceramides is highly reduced in the CerS2 null mouse; the inset shows C16-ceramide synthesis, which is unchanged. *, p 0.05; n 3.

Article Snippet: Fatty acyl-CoAs, ceramides, C6-NBD-sphingomyelin (SM), C6-NBD-glucosylceramide (GlcCer), C12-NBD-ceramide, C6-NBD-ceramide, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (Rho), and the internal standards for liquid chromatography electrospray ionization-tandem mass spectrometry (ESI-MS/MS) were purchased from Avanti Polar Lipids (Alabaster, AL).

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Figure Lengend Snippet: FIGURE 3. Ceramide and long chain base levels in CerS2 null mouse liver. Ceramide and sphinganine levels in CerS2 null mice versus age of the mice, measured by ESI-MS/MS. C24:1-ceramide is shown as an example of a very long acyl chain ceramide; data for the other very long acyl chain ceramides are in Table 1. For day 0, n 4; for other time points, n 2. *, p 0.05; **, p 0.01.

Article Snippet: Fatty acyl-CoAs, ceramides, C6-NBD-sphingomyelin (SM), C6-NBD-glucosylceramide (GlcCer), C12-NBD-ceramide, C6-NBD-ceramide, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (Rho), and the internal standards for liquid chromatography electrospray ionization-tandem mass spectrometry (ESI-MS/MS) were purchased from Avanti Polar Lipids (Alabaster, AL).

Techniques: Tandem Mass Spectroscopy

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Figure Lengend Snippet: Influence of tamoxifen and other agents on NBD-C6-ceramide metabolism in HL-60/VCR and in KG-1a cells. (A) TLC chromatogram illustrating qualitative effect of agents on production of NBD-C6-GC, NBD-hexanoic acid (denotes ceramidase activity), and NBD-C6-SM from NBD-C6-ceramide, in HL-60/VCR cells. NBD-C6-ceramide complexed to BSA was utilized as substrate, thus products are designated as NBD-containing. Whole cell incubations were carried out as detailed in Materials and methods. Note: Total incubation extracts (cells plus media) were applied to the origin of the TLC plate, thus the NBD-C6-cer spot is comprised of both extracellular and intracellular compound. Visualization was by fluorescence. (B) Quantitative analysis of NBD-C6-GC and NBD-C6-SM from the TLC chromatogram in panel (A). Quantitation was carried out as described in Materials and methods. (C) Effect of low-dose zosuquidar on NBD-C6-ceramide glycosylation in HL-60/VCR cells. Cells were exposed to the agent for 24 h before initiating the 2 h incubation with NBD-C6-ceramide. Cellular NBD-C6-GC was resolved by TLC and quantitated as described above. (D) Effect of zosuquidar on NBD-C6-ceramide metabolism in KG-1a cells. The 2 h incubations contained 100,000 and 25 μM substrate as detailed in Materials and methods. Data are presented as the mean ± SEM of three independent experiments in triplicate. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. Abbreviations: Tam, tamoxifen; DMT, N–desmethyltamoxifen; Zos, zosuquidar; Ver, verapamil; TØb, triphenylbutene; Ful, Fulvestrant; NBD-C6-cer, NBD-C6-ceramide; NBD-C6-GC, NBD-C6-glucosylceramide; NBD-hex, NBD-hexanoic acid; NBD-C6-SM, NBD-C6- sphingomyelin SEM, standard error of the mean

Article Snippet: C6-ceramide ( N -hexanoyl-D- erythro -sphingosine) and C6-NBD-lactosylceramide ( N -[6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]hexanoyl]-D-lactosyl-β1-1′-sphingosine) were obtained from Avanti Polar Lipids, Alabaster, AL. C6-NBD-ceramide ( N- [7-(4-nitrobenzo-2-oxa-1,3-diazole)]-6-aminocaproyl-D- erythro -sphingosine) was from Cayman Chemical Company, Ann Arbor, MI.

Techniques: Activity Assay, Incubation, Fluorescence, Quantitation Assay

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Figure Lengend Snippet: The effect of zosuquidar and tamoxifen on NBD-C6-ceramide metabolism, cytotoxicity, and ROS generation in the human colorectal cancer cell line LoVo. (A) TLC chromatogram, left; bar graph quantitation, right, showing effects of zosuquidar on synthesis of NBD-C6-GC and NBD-C6-SM from NBD-C6-ceramide. Cells, 100,000/reaction, were incubated for 2 h with 25 μM NBD-C6-ceramide substrate. Lipid extraction and TLC were conducted as described in Materials and methods. (B) Effect of zosuquidar on C6-ceramide cytotoxicity in LoVo cells. Cells (10,000/well) were seeded into 96-well plates in RPMI-1640 medium containing 10% FBS and the following day treated for 24 h with C6-ceramide and zosuquidar as indicated (formulated in RPMI-1640 medium containing 1% FBS). LoVo cell viability was determined by MTS reagent. (C) Assessment of drug regimen efficacy by CI and Fa isobologram, left; tabulated results of combination regimens 1–4, right. Results yielded CIs denoting synergism (CI= 0.3–0.7) and strong synergism (CI= 0.1–0.3). (D) Effect of tamoxifen and zosuquidar on NBD-C6-ceramide metabolism in LoVo cells. After a 2 h incubation, cell monolayers were rinsed free of extracellular NBD-C6-ceramide substrate followed by extraction of cellular lipids. (E) Effect of drug regimens on ROS generation. Mitochondrial superoxide was assayed using MitoSOX™ Red as described in Materials and methods. Cells were treated with the indicated agents for 18 h, and response was measured by fluorescence photomicrograph and spectroscopy. Left, photomicrographs denoting mitochondrial ROS levels (red fluorescence) in control and treated cells. Right, bar graph quantitation of ROS levels in control and treated cells after 18 h treatment with indicated agents. Concentrations (μM) of the agents used are noted in the bars. Data are presented as the mean ± SEM of three independent experiments in triplicate. ***P ≤ 0.001.

Article Snippet: C6-ceramide ( N -hexanoyl-D- erythro -sphingosine) and C6-NBD-lactosylceramide ( N -[6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]hexanoyl]-D-lactosyl-β1-1′-sphingosine) were obtained from Avanti Polar Lipids, Alabaster, AL. C6-NBD-ceramide ( N- [7-(4-nitrobenzo-2-oxa-1,3-diazole)]-6-aminocaproyl-D- erythro -sphingosine) was from Cayman Chemical Company, Ann Arbor, MI.

Techniques: Quantitation Assay, Incubation, Fluorescence, Spectroscopy