acid sphingomyelinase activity assay kit  (Echelon Biosciences)

Journal: PLoS ONE

Article Title: c-Abl Is an Upstream Regulator of Acid Sphingomyelinase in Apoptosis Induced by Inhibition of Integrins αvβ3 and αvβ5

doi: 10.1371/journal.pone.0042291

Figure Lengend Snippet: A ) Apoptosis (Apo-Direct kit) in ECV-304 cells treated with vehicle or RGDfV (5 µg/ml; 48 hrs). Left panel: representative flow cytometry plots showing percentage of apoptotic cells (FITC-dUTP positive cells in the two upper quadrants; dark blue dots). Color dots in the lower quadrants represent position in the cell cycle of the non-apoptotic cells based on DNA content (propidium iodide). Light blue is sub G 0 , green is G 1 , orange is S-phase and red is G 2 /M. Right panel: mean±SEM apoptotic (dark blue) cells of 24 independent experiments. B ) ASM activity (Echelon Biosciences kit) in extracts from ECV-304 treated with vehicle or RGDfV (5 µg/ml). Shown are mean±SEM of three independent experiments. C ) Real time quantitative RT-PCR for ASM from ECV-304 cells treated with vehicle or RGDfV (24 hrs). Shown are mean±SEM ASM mRNA normalized to GAPDH, depicted as fold-increase compared to baseline from 16 independent experiments performed in 1–4 replicates each. D–E ) Change in ceramide (D) and sphingomyelin (E) content in extracts from ECV-304 cells incubated with vehicle or RGDfV (5 µg/ml; 24 hrs) and analyzed by mass spectrometry. Bars represent mean±SEM fold change from control in one of two experiments with similar results, each of which was performed in 3 biological triplicates and analyzed in duplicates.

Article Snippet: ASM activity was determined using the ASM Assay Kit (#K-3200, Echelon Biosciences, Salt Lake City, UT) according to manufacturer’s instructions, using conditions that yielded readings in the linear range of the assay.

Techniques: Flow Cytometry, Activity Assay, Quantitative RT-PCR, Incubation, Mass Spectrometry

Journal: PLoS ONE

Article Title: c-Abl Is an Upstream Regulator of Acid Sphingomyelinase in Apoptosis Induced by Inhibition of Integrins αvβ3 and αvβ5

doi: 10.1371/journal.pone.0042291

Figure Lengend Snippet: ECV-304 cells were transfected with siRNA as described in . A ) Messenger RNA extracted from HBMEC 48 hrs after transfection with ASM1, ASM2, ERK, or control siRNA was analyzed for ASM expression by qRT-PCR (n = 6; p<0.001 for ASM1, p<0.001 for ASM2 and p = 0.54 for ERK by unpaired t-test for each siRNA compared to non-silencing control). B ) ASM activity in extracts of ECV-304 72 hrs after ASM-siRNA transfection compared to NC controls (Echelon Biosciences kit). Shown are mean±SEM; n = 3; p<0.001 for ASM1 and for ASM2, by unpaired t-test. C–D ) Change in ceramide (panel C) and sphingomyelin (panel D) in extracts of ECV-304 cells after NC, ASM siRNA #1 or ASM siRNA #2, that were then incubated with vehicle or RGDfV (5 µg/ml; 24 hrs) and analyzed by mass spectrometry. Bars represent mean±SEM fold change from control in one of two experiments with similar results, each of which was performed in total of 3 biological triplicates. Absolute lipid values are provided in .

Article Snippet: ASM activity was determined using the ASM Assay Kit (#K-3200, Echelon Biosciences, Salt Lake City, UT) according to manufacturer’s instructions, using conditions that yielded readings in the linear range of the assay.

Techniques: Transfection, Expressing, Quantitative RT-PCR, Activity Assay, Incubation, Mass Spectrometry

Journal: PLoS ONE

Article Title: c-Abl Is an Upstream Regulator of Acid Sphingomyelinase in Apoptosis Induced by Inhibition of Integrins αvβ3 and αvβ5

doi: 10.1371/journal.pone.0042291

Figure Lengend Snippet: A–B ) ECV-304 cells were treated with STI-571 (c-Abl inhibitor; 10 µM) or vehicle starting 2 hrs before adding RGDfV (5 µg/ml) or vehicle for additional 24 hrs. ( A ) ASM mRNA (real time qRT-PCR; mean±SEM of 4 independent experiments performed in 2 replicates), ( B ) ASM activity (Echelon Biosciences kit; mean±SEM of 2 independent experiments performed in 4 replicates). C–D ) ASM mRNA (real time qRT-PCR relative to GAPDH) of ECV-304 transfected with non-specific non-silencing negative control siRNA (NC) or c-Abl siRNA (48 hrs) and incubated with RGDfV (5 µg/ml) or vehicle for 24 hrs. Shown are mean±SEM, n = 4. Representative efficacy of c-Abl siRNA knockdown (western blot) is shown in Panel D. E ) Change in ceramide content in extracts from cells incubated with STI-571 or siRNA-c-Abl and their controls with/without RGDfV as in panels A–D was analyzed by mass spectrometry. Bars represent mean±SEM fold change in ceramides C 16 and C 18 from four (STI-571) and three (siRNA, either non-silencing controls NC, or c-Abl) independent experiments. Changes in other ceramides were not significant. P-values were calculated using paired t-tests. F–G ) Phospho-c-Abl (Y412) and total c-Abl (western blotting) in lysates of ECV-304 transfected with NC, ASM1 or ASM2 siRNA as in and treated 24 hrs with RGDfV (5 µg/ml) or vehicle. GAPDH served as loading control. Panel G shows a typical experiment and panel F shows mean±SEM of phospho-c-Abl (Y412) relative to total c-Abl from densitometry of three experiments. ASM mRNA level in these experiments was 28±1.2% of the non-silencing control samples for siRNA-ASM1 and 11.4±0.9% for siRNA-ASM2.

Article Snippet: ASM activity was determined using the ASM Assay Kit (#K-3200, Echelon Biosciences, Salt Lake City, UT) according to manufacturer’s instructions, using conditions that yielded readings in the linear range of the assay.

Techniques: Quantitative RT-PCR, Activity Assay, Transfection, Negative Control, Incubation, Western Blot, Mass Spectrometry

Journal: PLoS ONE

Article Title: c-Abl Is an Upstream Regulator of Acid Sphingomyelinase in Apoptosis Induced by Inhibition of Integrins αvβ3 and αvβ5

doi: 10.1371/journal.pone.0042291

Figure Lengend Snippet: Our data support a model in which inhibition of integrin αvβ3/αvβ5 signaling by RGDfV activates c-Abl (26). c-Abl in turn increases expression of ASM, which results in increased ASM activity and changes in sphingolipids, including increase in ceramides and decrease in sphingomyelins. The consequence of the increase in ASM expression and activity is apoptosis. Since ASM mediates the apoptosis, but it is not known which sphingolipid(s) is/are the mediator(s) of the apoptosis downstream of ASM, the downstream arrows are placed below ASM rather than below specific sphingolipids. Since downregulation of ASM expression leads to inhibition of caspase 3 and caspase 8 activity, caspases are placed downstream of ASM. Inhibition of c-Abl blocked the increase in ASM mRNA and activity and prevented the apoptosis, placing c-Abl upstream of ASM. It is likely that the interaction between c-Abl and ASM is not direct and that other yet-unknown mediators (denoted by the three small arrows and question mark) exist within this pathway. Not shown are possible initial upstream regulation of ASM by caspase 8 and other intermediate effectors.

Article Snippet: ASM activity was determined using the ASM Assay Kit (#K-3200, Echelon Biosciences, Salt Lake City, UT) according to manufacturer’s instructions, using conditions that yielded readings in the linear range of the assay.

Techniques: Inhibition, Expressing, Activity Assay

Journal: Cancer Cell International

Article Title: α-TEA-induced death receptor dependent apoptosis involves activation of acid sphingomyelinase and elevated ceramide-enriched cell surface membranes

doi: 10.1186/1475-2867-10-40

Figure Lengend Snippet: α-TEA induces ASMase activation . A, ASMase activity was detected using the Acid Sphingomyelinase Assay kit (Echelon) at different time points following treatment with 40 μM α-TEA. B, Cellular localization of ASMase following treatment with 40 μM α-TEA for 4 and 9 hrs (9 hrs vehicle treatment) was detected (green channel) using immunofluorescent staining of paraformaldehyde fixed cells. Arrows in α-TEA treated cells point to membrane ASMase versus arrows in vehicle control point to cellular dispersion of ASMase.

Article Snippet: ASMase activity was determined using an acid sphingomyelinase assay kit (Echelon Biosciences Inc. Salt Lake City, UT) following company's instructions.

Techniques: Activation Assay, Activity Assay, Staining

Journal: Cancer Cell International

Article Title: α-TEA-induced death receptor dependent apoptosis involves activation of acid sphingomyelinase and elevated ceramide-enriched cell surface membranes

doi: 10.1186/1475-2867-10-40

Figure Lengend Snippet: ASMase inhibitor desipramine blocks α-TEA-induced apoptosis . MDA-MB-231 cells were pre-treated with 12.5 μM desipramine for 2 hrs followed by treating the cells with 40 μM α-TEA for different time periods. A, Western blot analyses of cellular lysates were performed to detect cleavage of caspases-8 &-9 and PARP. B, ASMase activity was detected using the Acid Sphingomyelinase Assay kit (Echelon). C, Ceramide levels in cell surface membranes were detected by FACS analyses after reaction of living cells with primary ceramide antibody and fluorescence labeled secondary antibody after 4 hours of α-TEA treatment. Data are representative of three independent experiments. Data in B are presented as means ± S.D. of three independent experiments. (* = Significantly different, P < 0.05).

Article Snippet: ASMase activity was determined using an acid sphingomyelinase assay kit (Echelon Biosciences Inc. Salt Lake City, UT) following company's instructions.

Techniques: Western Blot, Activity Assay, Fluorescence, Labeling