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  • 96
    Millipore ceramide
    Exposure to C1P or <t>ceramide</t> induces P-glycoprotein transport activity at the blood-brain barrier. (A) Dose response of 20 minutes ceramide treatment, showing that ceramide increases specific P-glycoprotein activity in a concentration-dependent manner.
    Ceramide, supplied by Millipore, used in various techniques. Bioz Stars score: 96/100, based on 540 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 96 stars, based on 540 article reviews
    Price from $9.99 to $1999.99
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    88
    Tocris ceramide
    Sphingosine induces IL-1 release from LPS-primed macrophages. (A, B) LPS-treated (1 mg/mL, 2h) murine peritoneal macrophages were incubated for 1 h with the indicated concentration of (A) sphingosine (Sph) or (B) the Sph analogue FTY720. IL-1β in the supernatants was quantified by ELISA (top). Processing of pro-IL-1β (band at 31 to 17 kDa) was determined by western blot (bottom). (C) The structures of key mediators in the sphingolipid pathway are shown, and the structures of the Sph analogue FTY720, the S1P analogue FTY720-P and the sphingosine kinase inhibitor DMS are provided for comparison. (D, E) LPS-treated peritoneal macrophages were also incubated with the indicated concentrations of (D) <t>ceramide</t> or S1P or (E) 20 μM Sph, DMS, FTY720 and FTY720-P (FTYP) for 1h. IL-1β was quantified in supernatants by ELISA and pro-IL-1β processing to mature IL-1β was determined by western blot (E bottom). Data are shown as the mean 7 1 SD of three experiments. ***p
    Ceramide, supplied by Tocris, used in various techniques. Bioz Stars score: 88/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ceramide/product/Tocris
    Average 88 stars, based on 15 article reviews
    Price from $9.99 to $1999.99
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    94
    Millipore c2 ceramide
    Effects of high tau protein concentration on targeted migration of NSCs in the injured spinal cord of rats (Transwell assay). (A) Cresyl violet staining of NSCs in the control group and at different tau concentrations (0, 0.1, 0.5, 1.0 mg/mL) in the lower chamber (light microscope, × 100). (B) Cresyl violet staining of NSCs in the control, OA, <t>C2-ceramide</t> and unpretreated NSC groups (light microscope, × 100). (C) Optical density of cresyl violet staining of NSCs in the control group and at different tau concentrations (0, 0.1, 0.5, 1.0 mg/mL) in the lower chamber (no significant differences observed between tau concentrations). (D) Optical density of cresyl violet staining of NSCs in the control group, OA group, C2-ceramide group and unpretreated NSCs group (optical density was significantly lower in the OA and C2-ceramide groups than in the unpretreated NSCs group). * P
    C2 Ceramide, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 281 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 94 stars, based on 281 article reviews
    Price from $9.99 to $1999.99
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    94
    Avanti Polar ceramide
    CERT knockdown does not lead to a decrease in HexCer levels. A: Total sphingomyelin and HexCer levels of Hela cells treated with siRNA against the <t>ceramide</t> transport protein CERT. Scrambled (SCR) siRNA was used as control. B: Individual sphingomyelin
    Ceramide, supplied by Avanti Polar, used in various techniques. Bioz Stars score: 94/100, based on 450 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 94 stars, based on 450 article reviews
    Price from $9.99 to $1999.99
    ceramide - by Bioz Stars, 2020-08
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    Image Search Results


    Exposure to C1P or ceramide induces P-glycoprotein transport activity at the blood-brain barrier. (A) Dose response of 20 minutes ceramide treatment, showing that ceramide increases specific P-glycoprotein activity in a concentration-dependent manner.

    Journal: Molecular Pharmacology

    Article Title:

    doi: 10.1124/mol.116.107169

    Figure Lengend Snippet: Exposure to C1P or ceramide induces P-glycoprotein transport activity at the blood-brain barrier. (A) Dose response of 20 minutes ceramide treatment, showing that ceramide increases specific P-glycoprotein activity in a concentration-dependent manner.

    Article Snippet: Ceramide from bovine spinal cord, mouse monoclonal β -actin antibody, chlorpromazine hydrochloride, Ficoll PM 400, cycloheximide, SC-51089 hydrate [3-chloro- N ʹ-(3-pyridin-4-ylpropanoyl)-6 H -benzo[ b ][1,4]benzoxazepine-5-carbohydrazide;hydrate;hydrochloride], PF-04418948 [1-(4-fluorobenzoyl)-3-[(6-methoxynaphthalen-2-yl)oxymethyl]azetidine-3-carboxylic acid], celecoxib, NVP-231 [ N -(2-benzamido-1,3-benzothiazol-6-yl)adamantane-1-carboxamide], and all other chemicals were obtained from Sigma-Aldrich (St. Louis, MO).

    Techniques: Activity Assay, Concentration Assay

    Proposed signaling cascade for the induction of P-glycoprotein activity by ceramide and C1P.

    Journal: Molecular Pharmacology

    Article Title:

    doi: 10.1124/mol.116.107169

    Figure Lengend Snippet: Proposed signaling cascade for the induction of P-glycoprotein activity by ceramide and C1P.

    Article Snippet: Ceramide from bovine spinal cord, mouse monoclonal β -actin antibody, chlorpromazine hydrochloride, Ficoll PM 400, cycloheximide, SC-51089 hydrate [3-chloro- N ʹ-(3-pyridin-4-ylpropanoyl)-6 H -benzo[ b ][1,4]benzoxazepine-5-carbohydrazide;hydrate;hydrochloride], PF-04418948 [1-(4-fluorobenzoyl)-3-[(6-methoxynaphthalen-2-yl)oxymethyl]azetidine-3-carboxylic acid], celecoxib, NVP-231 [ N -(2-benzamido-1,3-benzothiazol-6-yl)adamantane-1-carboxamide], and all other chemicals were obtained from Sigma-Aldrich (St. Louis, MO).

    Techniques: Activity Assay

    Altered ceramide composition of 12R-LOX −/− epidermis. Free and ester-linked epidermal lipids were extracted and separated using TLC. Ceramides are classified as suggested in previous works ( Motta et al., 1993 ; Robson et al., 1994 ). B1–5 are lipids extracted after alkaline hydrolysis, most likely ester-linked epidermal lipids. (A) A representative TLC analysis of lipids extracted from wild-type and knockout epidermis. (B) Individual lipid levels quantified by densitometric analysis. Data are presented as the mean ± the SEM. n = 3. *, P

    Journal: The Journal of Cell Biology

    Article Title: 12R-lipoxygenase deficiency disrupts epidermal barrier function

    doi: 10.1083/jcb.200612116

    Figure Lengend Snippet: Altered ceramide composition of 12R-LOX −/− epidermis. Free and ester-linked epidermal lipids were extracted and separated using TLC. Ceramides are classified as suggested in previous works ( Motta et al., 1993 ; Robson et al., 1994 ). B1–5 are lipids extracted after alkaline hydrolysis, most likely ester-linked epidermal lipids. (A) A representative TLC analysis of lipids extracted from wild-type and knockout epidermis. (B) Individual lipid levels quantified by densitometric analysis. Data are presented as the mean ± the SEM. n = 3. *, P

    Article Snippet: Ceramide AS was purchased from Sigma-Aldrich.

    Techniques: Thin Layer Chromatography, Knock-Out

    The effect of TGF-β on ceramide-induced death of Hs578T breast cancer cells. Graphs in ( A , C and E ) represent per cent dead cells while ( B ) and ( D ) represents per cent of cell in pre-G1. Where ( A ) and ( B ) Hs578T cells were pre-incubated with IGFBP-3 (100 ng ml −1 ) for 24 h followed by a co-incubation of IGFBP-3 and C2 (where C2 > CT; P

    Journal: British Journal of Cancer

    Article Title: Differential interactions between IGFBP-3 and transforming growth factor-beta (TGF-?) in normal vs cancerous breast epithelial cells

    doi: 10.1038/sj.bjc.6600355

    Figure Lengend Snippet: The effect of TGF-β on ceramide-induced death of Hs578T breast cancer cells. Graphs in ( A , C and E ) represent per cent dead cells while ( B ) and ( D ) represents per cent of cell in pre-G1. Where ( A ) and ( B ) Hs578T cells were pre-incubated with IGFBP-3 (100 ng ml −1 ) for 24 h followed by a co-incubation of IGFBP-3 and C2 (where C2 > CT; P

    Article Snippet: The ceramide analogue, C2, was purchased from Calbiochem (Nottingham, UK).

    Techniques: Incubation

    The effects of IGFBP-3 and TGF-β on ceramide-induced death of the MCF-10A cells. Graphs ( A + B ) represent the per cent of dead cells. MCF-10A cells were ( A ) pre-incubated with IGFBP-3 (100 ng ml −1 ) for 24 h followed by a co-incubation with an apoptotic dose of C2 for a further 24 h (where ***C2 > CT; P

    Journal: British Journal of Cancer

    Article Title: Differential interactions between IGFBP-3 and transforming growth factor-beta (TGF-?) in normal vs cancerous breast epithelial cells

    doi: 10.1038/sj.bjc.6600355

    Figure Lengend Snippet: The effects of IGFBP-3 and TGF-β on ceramide-induced death of the MCF-10A cells. Graphs ( A + B ) represent the per cent of dead cells. MCF-10A cells were ( A ) pre-incubated with IGFBP-3 (100 ng ml −1 ) for 24 h followed by a co-incubation with an apoptotic dose of C2 for a further 24 h (where ***C2 > CT; P

    Article Snippet: The ceramide analogue, C2, was purchased from Calbiochem (Nottingham, UK).

    Techniques: Incubation

    Identification of novel ceramide analogs that sensitize human colon carcinoma cells to FasL-induced apoptosis. ( A ) Human colon carcinoma SW480, RKO and HCT116 cells were cultured in the presence of the indicated ceramide analogs (10 μM), with or without MegaFasL (SW480 = 25 ng/ml, RKO = 50 ng/ml, and HCT116 = 10 ng/ml) for approximately 24 h. Both floating and adherent cells were harvested, stained with Annexin V and PI, and analyzed by flow cytometry. Shown are representative plots of apoptotic cell death. ( B ) Quantification of apoptotic cell death. % apoptotic cell death was calculated as % Annexin V + PI + cells in the presence of ceramide analogs plus MegaFasL - % Annexin V + PI + cells in the control group. Column: mean; Bar: SD.

    Journal: Scientific Reports

    Article Title: Ceramide mediates FasL-induced caspase 8 activation in colon carcinoma cells to enhance FasL-induced cytotoxicity by tumor-specific cytotoxic T lymphocytes

    doi: 10.1038/srep30816

    Figure Lengend Snippet: Identification of novel ceramide analogs that sensitize human colon carcinoma cells to FasL-induced apoptosis. ( A ) Human colon carcinoma SW480, RKO and HCT116 cells were cultured in the presence of the indicated ceramide analogs (10 μM), with or without MegaFasL (SW480 = 25 ng/ml, RKO = 50 ng/ml, and HCT116 = 10 ng/ml) for approximately 24 h. Both floating and adherent cells were harvested, stained with Annexin V and PI, and analyzed by flow cytometry. Shown are representative plots of apoptotic cell death. ( B ) Quantification of apoptotic cell death. % apoptotic cell death was calculated as % Annexin V + PI + cells in the presence of ceramide analogs plus MegaFasL - % Annexin V + PI + cells in the control group. Column: mean; Bar: SD.

    Article Snippet: Briefly, tumor cells were cultured in the presence of the indicated ceramide analogs or ceramide analogs plus MegaFasL for 4 h. Cells were collected and lysed in cytosol buffer [10 mM Hepes, pH 7.4, 250 mM Sucrose, 70 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, protease and phosphatase inhibitor cocktails (Calbiochem, Billerica, MA), and 0.01% digitonin] for 10 min. Cytosolic fractions were resolved in 4–20% SDS polyacrylamide gel and analyzed by Western blotting.

    Techniques: Cell Culture, Staining, Flow Cytometry, Cytometry

    Identification of novel ceramide analogs that increase efficacy of tumor-specific CTL-mediated tumor lysis. ( A ) Mouse colon carcinoma CT26 cells were labeled with CFSE and seeded in U-bottom 96-well plates. Tumor-specific perforin-deficient pfp CTLs were then added to the tumor cultures at the indicated effector/tumor ratios (E/T ratios) and cultured for approximately 24 h. CTL and tumor culture mixtures were harvested and stained with PI and analyzed by flow cytometry. Shown are representative plots. ( B ) Quantification of CTL-induced tumor cell death kinetics. Cells as shown in A were gated for CFSE + tumor cells. The gated cells were then analyzed for PI + cells. % tumor cell lysis was calculated as % CFSE + PI + cells in the presence of CTLs - % CFSE + PI + cells in the absence of CTLs. ( C ) CT26 cells were labeled with CFSE as in A and cultured in the presence of the indicated ceramide analogs (10 μM) without (top panel) or with (bottom panel) pfpCTLs for approximately 24 h. CTL-induced tumor cell death was analyzed as in A. ( D ) Quantification of CTL-induced tumor cell death in the absence or presence of ceramide analogs. Cells as shown in C were gated for CFSE + tumor cells. The gated cells were then analyzed for PI + cells. % tumor cell lysis was calculated as % CFSE + PI + cells in the presence of ceramide analogs or ceramide analogs plus pfp CTLs - % CFSE + PI + cells in the absence of ceramide analogs or ceramide analogs plus pfp CTLs. Column: mean; Bar: SD. Red **indicated p

    Journal: Scientific Reports

    Article Title: Ceramide mediates FasL-induced caspase 8 activation in colon carcinoma cells to enhance FasL-induced cytotoxicity by tumor-specific cytotoxic T lymphocytes

    doi: 10.1038/srep30816

    Figure Lengend Snippet: Identification of novel ceramide analogs that increase efficacy of tumor-specific CTL-mediated tumor lysis. ( A ) Mouse colon carcinoma CT26 cells were labeled with CFSE and seeded in U-bottom 96-well plates. Tumor-specific perforin-deficient pfp CTLs were then added to the tumor cultures at the indicated effector/tumor ratios (E/T ratios) and cultured for approximately 24 h. CTL and tumor culture mixtures were harvested and stained with PI and analyzed by flow cytometry. Shown are representative plots. ( B ) Quantification of CTL-induced tumor cell death kinetics. Cells as shown in A were gated for CFSE + tumor cells. The gated cells were then analyzed for PI + cells. % tumor cell lysis was calculated as % CFSE + PI + cells in the presence of CTLs - % CFSE + PI + cells in the absence of CTLs. ( C ) CT26 cells were labeled with CFSE as in A and cultured in the presence of the indicated ceramide analogs (10 μM) without (top panel) or with (bottom panel) pfpCTLs for approximately 24 h. CTL-induced tumor cell death was analyzed as in A. ( D ) Quantification of CTL-induced tumor cell death in the absence or presence of ceramide analogs. Cells as shown in C were gated for CFSE + tumor cells. The gated cells were then analyzed for PI + cells. % tumor cell lysis was calculated as % CFSE + PI + cells in the presence of ceramide analogs or ceramide analogs plus pfp CTLs - % CFSE + PI + cells in the absence of ceramide analogs or ceramide analogs plus pfp CTLs. Column: mean; Bar: SD. Red **indicated p

    Article Snippet: Briefly, tumor cells were cultured in the presence of the indicated ceramide analogs or ceramide analogs plus MegaFasL for 4 h. Cells were collected and lysed in cytosol buffer [10 mM Hepes, pH 7.4, 250 mM Sucrose, 70 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, protease and phosphatase inhibitor cocktails (Calbiochem, Billerica, MA), and 0.01% digitonin] for 10 min. Cytosolic fractions were resolved in 4–20% SDS polyacrylamide gel and analyzed by Western blotting.

    Techniques: CTL Assay, Lysis, Labeling, Cell Culture, Staining, Flow Cytometry, Cytometry

    Fas mediates activation of caspase 8 and MAPK and ceramide analogs enhance FasL-induced caspase 8 activation. ( A ) Human colon carcinoma SW480, RKO, and HCT116 cells were cultured in the presence of the indicated ceramide analogs or ceramide analogs plus MegaFasL for 4 h. Cells were collected and lysed in cytosol buffer. Cytosolic fractions were resolved in 4–20% SDS polyacrylamide gel and analyzed by Western blotting using anti-active caspase 8 and anti-cleaved PARP antibodies, respectively. The membranes were stripped and re-probed with anti-β-actin antibody. The pro-caspase 8, cleaved caspase 8, cleaved PARP and β-actin are indicated at the right. The locations of molecular weight markers are indicated at the left. ( B ) SW480 and RKO cells were cultured in the presence of FasL (100 ng/ml), IG7 (10 μM), or both FasL and IG7 for 24 hours and total lysate was prepared from the treated cells. Total lysate was analyzed by Western blotting as in A with antibodies that are specific for the indicated proteins. ( C , D ) SW480 ( C ) and RKO ( D ) cells were cultured in the presence of FasL (50 ng/ml), IG7 (10 μM) or both FasL and IG7 for 1 and 3 hours. Cytosol fractions were isolated as in A and analyzed by Western blotting as in A with antibodies that are specific for the indicated proteins.

    Journal: Scientific Reports

    Article Title: Ceramide mediates FasL-induced caspase 8 activation in colon carcinoma cells to enhance FasL-induced cytotoxicity by tumor-specific cytotoxic T lymphocytes

    doi: 10.1038/srep30816

    Figure Lengend Snippet: Fas mediates activation of caspase 8 and MAPK and ceramide analogs enhance FasL-induced caspase 8 activation. ( A ) Human colon carcinoma SW480, RKO, and HCT116 cells were cultured in the presence of the indicated ceramide analogs or ceramide analogs plus MegaFasL for 4 h. Cells were collected and lysed in cytosol buffer. Cytosolic fractions were resolved in 4–20% SDS polyacrylamide gel and analyzed by Western blotting using anti-active caspase 8 and anti-cleaved PARP antibodies, respectively. The membranes were stripped and re-probed with anti-β-actin antibody. The pro-caspase 8, cleaved caspase 8, cleaved PARP and β-actin are indicated at the right. The locations of molecular weight markers are indicated at the left. ( B ) SW480 and RKO cells were cultured in the presence of FasL (100 ng/ml), IG7 (10 μM), or both FasL and IG7 for 24 hours and total lysate was prepared from the treated cells. Total lysate was analyzed by Western blotting as in A with antibodies that are specific for the indicated proteins. ( C , D ) SW480 ( C ) and RKO ( D ) cells were cultured in the presence of FasL (50 ng/ml), IG7 (10 μM) or both FasL and IG7 for 1 and 3 hours. Cytosol fractions were isolated as in A and analyzed by Western blotting as in A with antibodies that are specific for the indicated proteins.

    Article Snippet: Briefly, tumor cells were cultured in the presence of the indicated ceramide analogs or ceramide analogs plus MegaFasL for 4 h. Cells were collected and lysed in cytosol buffer [10 mM Hepes, pH 7.4, 250 mM Sucrose, 70 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, protease and phosphatase inhibitor cocktails (Calbiochem, Billerica, MA), and 0.01% digitonin] for 10 min. Cytosolic fractions were resolved in 4–20% SDS polyacrylamide gel and analyzed by Western blotting.

    Techniques: Activation Assay, Cell Culture, Western Blot, Molecular Weight, Isolation

    Ceramide analog enhances FasL-induced apoptosis in tumor cells in a Fas-dependent mechanism. ( A ) Tumor cells were treated as shown in the absence or presence of ceramide analog IG7 (10 μM) for approximately 24 h. Both floating and adherent cells were collected and stained with PI and Annexin V. Cells were then analyzed by flow cytometry. ( B ) Cells as shown in A are quantified for apoptosis. Percent apoptotic cell death was calculated as (% Annexin V + PI + cells of treated cells) − (% Annexin V + PI + cells in the absence of FasL). Column: mean; Bar: SD.

    Journal: Scientific Reports

    Article Title: Ceramide mediates FasL-induced caspase 8 activation in colon carcinoma cells to enhance FasL-induced cytotoxicity by tumor-specific cytotoxic T lymphocytes

    doi: 10.1038/srep30816

    Figure Lengend Snippet: Ceramide analog enhances FasL-induced apoptosis in tumor cells in a Fas-dependent mechanism. ( A ) Tumor cells were treated as shown in the absence or presence of ceramide analog IG7 (10 μM) for approximately 24 h. Both floating and adherent cells were collected and stained with PI and Annexin V. Cells were then analyzed by flow cytometry. ( B ) Cells as shown in A are quantified for apoptosis. Percent apoptotic cell death was calculated as (% Annexin V + PI + cells of treated cells) − (% Annexin V + PI + cells in the absence of FasL). Column: mean; Bar: SD.

    Article Snippet: Briefly, tumor cells were cultured in the presence of the indicated ceramide analogs or ceramide analogs plus MegaFasL for 4 h. Cells were collected and lysed in cytosol buffer [10 mM Hepes, pH 7.4, 250 mM Sucrose, 70 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, protease and phosphatase inhibitor cocktails (Calbiochem, Billerica, MA), and 0.01% digitonin] for 10 min. Cytosolic fractions were resolved in 4–20% SDS polyacrylamide gel and analyzed by Western blotting.

    Techniques: Staining, Flow Cytometry, Cytometry

    FasL induces NF-κB activation and ceramide analog increases FasL-induced NF-κB activation. SW480 and RKO cells were treated with FasL (50 ng/ml), IG7 (10 μM), or FasL and IG7 for 1h and nuclear extracts were prepared from the tumor cells and analyzed for canonical NF-κB activity using EMSA with NF-κB consensus sequence-containing DNA probe as described in the materials and methods. Anti-p65 and anti-p50 antibodies were used to identify the canonical NF-κB-DNA complexes. The DNA-NF-κB complexes are indicated at the right.

    Journal: Scientific Reports

    Article Title: Ceramide mediates FasL-induced caspase 8 activation in colon carcinoma cells to enhance FasL-induced cytotoxicity by tumor-specific cytotoxic T lymphocytes

    doi: 10.1038/srep30816

    Figure Lengend Snippet: FasL induces NF-κB activation and ceramide analog increases FasL-induced NF-κB activation. SW480 and RKO cells were treated with FasL (50 ng/ml), IG7 (10 μM), or FasL and IG7 for 1h and nuclear extracts were prepared from the tumor cells and analyzed for canonical NF-κB activity using EMSA with NF-κB consensus sequence-containing DNA probe as described in the materials and methods. Anti-p65 and anti-p50 antibodies were used to identify the canonical NF-κB-DNA complexes. The DNA-NF-κB complexes are indicated at the right.

    Article Snippet: Briefly, tumor cells were cultured in the presence of the indicated ceramide analogs or ceramide analogs plus MegaFasL for 4 h. Cells were collected and lysed in cytosol buffer [10 mM Hepes, pH 7.4, 250 mM Sucrose, 70 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, protease and phosphatase inhibitor cocktails (Calbiochem, Billerica, MA), and 0.01% digitonin] for 10 min. Cytosolic fractions were resolved in 4–20% SDS polyacrylamide gel and analyzed by Western blotting.

    Techniques: Activation Assay, Activity Assay, Sequencing

    Pharmacological restoration of ASM to the normal range improves pathology in AD mice. (A) Protocol of AMI treatment in APP/PS1 mice. (B) ASM was estimated in the blood plasma ( n = 12–14 per group) and brain ( n = 9–10 per group) of APP/PS1 mice after AMI treatment. (C) Sphingomyelin, ceramide, and AC were determined using UPLC based methods in the plasma ( n = 9 per group) and brain ( n = 8 per group). (D) Mice brain sections were stained with thioflavin S to detect Aβ (bars, 200 µm). The relative area occupied by Aβ plaques were determined ( n = 6 per group). (E–G) Aβ40 and Aβ42 in the brains of AMI treated or nontreated APP/PS1 mice were assessed using immunofluorescence staining (E and F; n = 8 per group; bars, 200 µm) and ELISA kits (G; n = 6 per group). (H and I) Western blot analyses and quantification for LC3, Beclin-1, p62, cathepsin D, TFEB, and Lamp1 in the brains of APP/PS1 mice treated with AMI or control ( n = 6–8 per group). (J) Cathepsin D activity in the brain extracts of AMI-treated or nontreated APP/PS1 mice ( n = 4 per group). (K) Escape latencies of APP/PS1 mice treated with AMI or control over 10 d (WT, n = 14; nontreated APP/PS1, n = 10; and AMI-treated APP/PS1, n = 12). (L–O) Probe trial day 11. (L and M) Path length (L) and swim speed (M) were recorded and analyzed. (N) Time spent in target platform and other quadrants was measured. (O) The number of times each animal entered the small target zone during the 60-s probe trial. (P) Representative swimming paths at day 10 of training. Data are representative three independent experiments. B–J and N, Student’s t test; K–M and O, one-way ANOVA, Tukey’s post hoc test. *, P

    Journal: The Journal of Experimental Medicine

    Article Title: Acid sphingomyelinase modulates the autophagic process by controlling lysosomal biogenesis in Alzheimer’s disease

    doi: 10.1084/jem.20132451

    Figure Lengend Snippet: Pharmacological restoration of ASM to the normal range improves pathology in AD mice. (A) Protocol of AMI treatment in APP/PS1 mice. (B) ASM was estimated in the blood plasma ( n = 12–14 per group) and brain ( n = 9–10 per group) of APP/PS1 mice after AMI treatment. (C) Sphingomyelin, ceramide, and AC were determined using UPLC based methods in the plasma ( n = 9 per group) and brain ( n = 8 per group). (D) Mice brain sections were stained with thioflavin S to detect Aβ (bars, 200 µm). The relative area occupied by Aβ plaques were determined ( n = 6 per group). (E–G) Aβ40 and Aβ42 in the brains of AMI treated or nontreated APP/PS1 mice were assessed using immunofluorescence staining (E and F; n = 8 per group; bars, 200 µm) and ELISA kits (G; n = 6 per group). (H and I) Western blot analyses and quantification for LC3, Beclin-1, p62, cathepsin D, TFEB, and Lamp1 in the brains of APP/PS1 mice treated with AMI or control ( n = 6–8 per group). (J) Cathepsin D activity in the brain extracts of AMI-treated or nontreated APP/PS1 mice ( n = 4 per group). (K) Escape latencies of APP/PS1 mice treated with AMI or control over 10 d (WT, n = 14; nontreated APP/PS1, n = 10; and AMI-treated APP/PS1, n = 12). (L–O) Probe trial day 11. (L and M) Path length (L) and swim speed (M) were recorded and analyzed. (N) Time spent in target platform and other quadrants was measured. (O) The number of times each animal entered the small target zone during the 60-s probe trial. (P) Representative swimming paths at day 10 of training. Data are representative three independent experiments. B–J and N, Student’s t test; K–M and O, one-way ANOVA, Tukey’s post hoc test. *, P

    Article Snippet: To quantify the sphingomyelin and ceramide levels, the dried lipid extract was resuspended in 0.2% Igepal CA-630 (Sigma-Aldrich) and the levels of each lipid were determined using the UPLC system.

    Techniques: Mouse Assay, Staining, Immunofluorescence, Enzyme-linked Immunosorbent Assay, Western Blot, Activity Assay

    Partial genetic inhibition of ASM leads to decreased AD pathology in the APP/PS1 mice. (A) Generation of the APP/PS1/ ASM +/− mice. (B) Body weights of WT, APP/PS1, ASM +/− , and APP/PS1/ ASM +/− mice were determined at 9 mo of age ( n = 14 per group). (C) ASM activity in blood plasma ( n = 14–15 per group), brain ( n = 13–14 per group), and fibroblast ( n = 8 per group) derived from WT, APP/PS1, ASM +/− , and APP/PS1/ ASM +/− mice. (D) ASM activity was assessed in neuron and microglia isolated from mouse brain (WT, n = 8; APP/PS1, n = 6; and APP/PS1/ ASM +/− , n = 6). (E) Detection of sphingomyelin, ceramide, and AC in plasma ( n = 8–10 per group), brain ( n = 7–9 per group), and tail ( n = 5–6 per group) fibroblast. (F) Mice brain sections were stained with thioflavin S in APP/PS1 and APP/PS1/ ASM +/− mice. The relative area occupied by Aβ plaques were determined ( n = 6–7 per group; bars, 100 µm). (G–I) Analysis of Aβ40 and Aβ42 depositions from the mice brain samples using immunofluorescence staining (G and H; n = 6–7 per group; bars, 200 µm) and ELISA kits (I; n = 8 per group). (J and K) Confocal laser microscope images and quantification of cerebral amyloid angiopathy (J; n = 6 per group; bars, 50 µm) and tau hyperphosphorylation (K; n = 6 per group; bars, 20 µm) in APP/PS1 and APP/PS1/ ASM +/− mice. Data are representative of two (D and K), three (B, C, and E), or four (F–J) independent experiments. B–E, one-way ANOVA, Tukey’s post hoc test. F–K, Student’s t test. *, P

    Journal: The Journal of Experimental Medicine

    Article Title: Acid sphingomyelinase modulates the autophagic process by controlling lysosomal biogenesis in Alzheimer’s disease

    doi: 10.1084/jem.20132451

    Figure Lengend Snippet: Partial genetic inhibition of ASM leads to decreased AD pathology in the APP/PS1 mice. (A) Generation of the APP/PS1/ ASM +/− mice. (B) Body weights of WT, APP/PS1, ASM +/− , and APP/PS1/ ASM +/− mice were determined at 9 mo of age ( n = 14 per group). (C) ASM activity in blood plasma ( n = 14–15 per group), brain ( n = 13–14 per group), and fibroblast ( n = 8 per group) derived from WT, APP/PS1, ASM +/− , and APP/PS1/ ASM +/− mice. (D) ASM activity was assessed in neuron and microglia isolated from mouse brain (WT, n = 8; APP/PS1, n = 6; and APP/PS1/ ASM +/− , n = 6). (E) Detection of sphingomyelin, ceramide, and AC in plasma ( n = 8–10 per group), brain ( n = 7–9 per group), and tail ( n = 5–6 per group) fibroblast. (F) Mice brain sections were stained with thioflavin S in APP/PS1 and APP/PS1/ ASM +/− mice. The relative area occupied by Aβ plaques were determined ( n = 6–7 per group; bars, 100 µm). (G–I) Analysis of Aβ40 and Aβ42 depositions from the mice brain samples using immunofluorescence staining (G and H; n = 6–7 per group; bars, 200 µm) and ELISA kits (I; n = 8 per group). (J and K) Confocal laser microscope images and quantification of cerebral amyloid angiopathy (J; n = 6 per group; bars, 50 µm) and tau hyperphosphorylation (K; n = 6 per group; bars, 20 µm) in APP/PS1 and APP/PS1/ ASM +/− mice. Data are representative of two (D and K), three (B, C, and E), or four (F–J) independent experiments. B–E, one-way ANOVA, Tukey’s post hoc test. F–K, Student’s t test. *, P

    Article Snippet: To quantify the sphingomyelin and ceramide levels, the dried lipid extract was resuspended in 0.2% Igepal CA-630 (Sigma-Aldrich) and the levels of each lipid were determined using the UPLC system.

    Techniques: Inhibition, Mouse Assay, Activity Assay, Derivative Assay, Isolation, Staining, Immunofluorescence, Enzyme-linked Immunosorbent Assay, Microscopy

    ASM is increased in AD and complete ASM gene deficiency exacerbates pathology of APP/PS1 mice. (A and B) ASM was estimated in the blood plasma (A; control, n = 30; AD, n = 40; and PD, n = 20) and fibroblast (B; control, n = 24; PS1-FAD, n = 24; ApoE4, n = 24; and PD, n = 12) with AD, PD, or normal controls. (C) ASM activity did not show passage differences between AD and normal fibroblasts ( n = 8 per passage group). (D) Detection of sphingomyelin, ceramide, and AC in plasma (control, n = 20–22; and AD, n = 33–35) and fibroblast (control, n = 12; PS1-FAD, n = 18; and ApoE4, n = 18). (E) Crossing scheme to generate WT, APP/PS1, ASM −/− , and APP/PS1/ ASM −/− mice. PCR-based genotyping to detect WT, APP/PS1, ASM −/− , and APP/PS1/ ASM −/− mice. (F) Survival curves of WT ( n = 26), APP/PS1 ( n = 30), ASM −/− ( n = 30), and APP/PS1/ ASM −/− ( n = 25) mice. (G) Body weights of WT, APP/PS1, ASM −/− , and APP/PS1/ ASM −/− mice were determined at the indicated ages ( n = 6–7 per group). (H–J) Brain sections from 7-mo-old mice were immunostained with anti–active caspase3 (H; n = 4 per group; bars, 50 µm), anti-GFAP (I; n = 4 per group; bars, 100 µm), and anti–Iba-1 (J; n = 4 per group; bars, 100 µm). Data are representative of three independent experiments. A–D and G, Student’s t test. H–J, one-way ANOVA, Tukey’s post hoc test. *, P

    Journal: The Journal of Experimental Medicine

    Article Title: Acid sphingomyelinase modulates the autophagic process by controlling lysosomal biogenesis in Alzheimer’s disease

    doi: 10.1084/jem.20132451

    Figure Lengend Snippet: ASM is increased in AD and complete ASM gene deficiency exacerbates pathology of APP/PS1 mice. (A and B) ASM was estimated in the blood plasma (A; control, n = 30; AD, n = 40; and PD, n = 20) and fibroblast (B; control, n = 24; PS1-FAD, n = 24; ApoE4, n = 24; and PD, n = 12) with AD, PD, or normal controls. (C) ASM activity did not show passage differences between AD and normal fibroblasts ( n = 8 per passage group). (D) Detection of sphingomyelin, ceramide, and AC in plasma (control, n = 20–22; and AD, n = 33–35) and fibroblast (control, n = 12; PS1-FAD, n = 18; and ApoE4, n = 18). (E) Crossing scheme to generate WT, APP/PS1, ASM −/− , and APP/PS1/ ASM −/− mice. PCR-based genotyping to detect WT, APP/PS1, ASM −/− , and APP/PS1/ ASM −/− mice. (F) Survival curves of WT ( n = 26), APP/PS1 ( n = 30), ASM −/− ( n = 30), and APP/PS1/ ASM −/− ( n = 25) mice. (G) Body weights of WT, APP/PS1, ASM −/− , and APP/PS1/ ASM −/− mice were determined at the indicated ages ( n = 6–7 per group). (H–J) Brain sections from 7-mo-old mice were immunostained with anti–active caspase3 (H; n = 4 per group; bars, 50 µm), anti-GFAP (I; n = 4 per group; bars, 100 µm), and anti–Iba-1 (J; n = 4 per group; bars, 100 µm). Data are representative of three independent experiments. A–D and G, Student’s t test. H–J, one-way ANOVA, Tukey’s post hoc test. *, P

    Article Snippet: To quantify the sphingomyelin and ceramide levels, the dried lipid extract was resuspended in 0.2% Igepal CA-630 (Sigma-Aldrich) and the levels of each lipid were determined using the UPLC system.

    Techniques: Mouse Assay, Activity Assay, Polymerase Chain Reaction

    Selective elevation of diunsaturated ceramides requires BAX and BAK. A: Extracted ion chromatograms of C16-C24 ceramides. m/z expansion: ±50 ppm. B: Quantification of C16-C24 ceramides with d 31 -d18:1/16:0 ceramide as internal standard. C: Total mitochondrial di/monounsaturated ceramide ratios for WT and DKO MEFs. D: WT and DKO MEF mitochondrial di/monounsaturated ceramide ratio by N-acyl chain. * P ≤ 0.05, ** P ≤ 0.01, and *** P ≤ 0.001, two-tailed Student’s t -test (SEM; n = 3 for all sections).

    Journal: Journal of Lipid Research

    Article Title: Regulation of mitochondrial ceramide distribution by members of the BCL-2 family [S]

    doi: 10.1194/jlr.M058750

    Figure Lengend Snippet: Selective elevation of diunsaturated ceramides requires BAX and BAK. A: Extracted ion chromatograms of C16-C24 ceramides. m/z expansion: ±50 ppm. B: Quantification of C16-C24 ceramides with d 31 -d18:1/16:0 ceramide as internal standard. C: Total mitochondrial di/monounsaturated ceramide ratios for WT and DKO MEFs. D: WT and DKO MEF mitochondrial di/monounsaturated ceramide ratio by N-acyl chain. * P ≤ 0.05, ** P ≤ 0.01, and *** P ≤ 0.001, two-tailed Student’s t -test (SEM; n = 3 for all sections).

    Article Snippet: Chemicals for ceramide synthesis were as follows: Grubbs second-generation catalyst, N-succinimidyl palmitate, palmitoleic acid, triethylamine, and vinyl magnesium bromide (Sigma-Aldrich); ( S )- Garner aldehyde (TCI America); oct-7-enal (Novel Chemical Solutions); heptyltriphenylphosphonium bromide, sodium bis (trimethylsilyl)amide, and oxalyl chloride (Alfa Aesar); d18:1 sphingosine (Avanti Polar Lipids); tetrahydrofuran (Acros Organics); and pyridine (Mallinckrodt Chemicals).

    Techniques: Two Tailed Test

    ). B: Generation of d 12 -d18:2/16:0 ceramide by d 14 -palmitoleic acid-treated MEFs.

    Journal: Journal of Lipid Research

    Article Title: Regulation of mitochondrial ceramide distribution by members of the BCL-2 family [S]

    doi: 10.1194/jlr.M058750

    Figure Lengend Snippet: ). B: Generation of d 12 -d18:2/16:0 ceramide by d 14 -palmitoleic acid-treated MEFs.

    Article Snippet: Chemicals for ceramide synthesis were as follows: Grubbs second-generation catalyst, N-succinimidyl palmitate, palmitoleic acid, triethylamine, and vinyl magnesium bromide (Sigma-Aldrich); ( S )- Garner aldehyde (TCI America); oct-7-enal (Novel Chemical Solutions); heptyltriphenylphosphonium bromide, sodium bis (trimethylsilyl)amide, and oxalyl chloride (Alfa Aesar); d18:1 sphingosine (Avanti Polar Lipids); tetrahydrofuran (Acros Organics); and pyridine (Mallinckrodt Chemicals).

    Techniques:

    Additional sphingolipids and ceramide levels in WT, DKO, and SKO MEFs. A: Relative sphingomyelin levels in WT and DKO MEF mitochondria. No statistically significant changes were observed. B: Relative sphingadiene, sphingosine, and sphinganine levels in WT and DKO MEF mitochondria. No statistically significant changes were observed. C: Relative C16-C24 ceramide levels in WT and Bax −/− MEF mitochondria. D: Relative C16-C24 ceramide levels in WT and Bak −/− MEF mitochondria. * P ≤ 0.05 and ** P ≤ 0.01, two-tailed Student’s t -test (SEM; n = 3 for C and D).

    Journal: Journal of Lipid Research

    Article Title: Regulation of mitochondrial ceramide distribution by members of the BCL-2 family [S]

    doi: 10.1194/jlr.M058750

    Figure Lengend Snippet: Additional sphingolipids and ceramide levels in WT, DKO, and SKO MEFs. A: Relative sphingomyelin levels in WT and DKO MEF mitochondria. No statistically significant changes were observed. B: Relative sphingadiene, sphingosine, and sphinganine levels in WT and DKO MEF mitochondria. No statistically significant changes were observed. C: Relative C16-C24 ceramide levels in WT and Bax −/− MEF mitochondria. D: Relative C16-C24 ceramide levels in WT and Bak −/− MEF mitochondria. * P ≤ 0.05 and ** P ≤ 0.01, two-tailed Student’s t -test (SEM; n = 3 for C and D).

    Article Snippet: Chemicals for ceramide synthesis were as follows: Grubbs second-generation catalyst, N-succinimidyl palmitate, palmitoleic acid, triethylamine, and vinyl magnesium bromide (Sigma-Aldrich); ( S )- Garner aldehyde (TCI America); oct-7-enal (Novel Chemical Solutions); heptyltriphenylphosphonium bromide, sodium bis (trimethylsilyl)amide, and oxalyl chloride (Alfa Aesar); d18:1 sphingosine (Avanti Polar Lipids); tetrahydrofuran (Acros Organics); and pyridine (Mallinckrodt Chemicals).

    Techniques: Two Tailed Test

    BAX and BAK selectively regulate d18:2-Cers in MEFs and iBMKs. A: WT and DKO MEF mitochondrial and whole cell d18:2-Cer/d18:1-Cer ratio by N-acyl chain. B: WT and DKO iBMK mitochondrial and whole cell d18:2-Cer/d18:1-Cer ratio by N-acyl chain. C: Total d18:1/X:0, d18:2/X:0, and d18:1/X:1 ceramide levels in WT and DKO MEFs; data are normalized to WT d18:1/X:0 value. * P ≤ 0.05, ** P ≤ 0.01, and *** P ≤ 0.001, two-tailed Student’s t -test (SEM; n = 4 for all sections).

    Journal: Journal of Lipid Research

    Article Title: Regulation of mitochondrial ceramide distribution by members of the BCL-2 family [S]

    doi: 10.1194/jlr.M058750

    Figure Lengend Snippet: BAX and BAK selectively regulate d18:2-Cers in MEFs and iBMKs. A: WT and DKO MEF mitochondrial and whole cell d18:2-Cer/d18:1-Cer ratio by N-acyl chain. B: WT and DKO iBMK mitochondrial and whole cell d18:2-Cer/d18:1-Cer ratio by N-acyl chain. C: Total d18:1/X:0, d18:2/X:0, and d18:1/X:1 ceramide levels in WT and DKO MEFs; data are normalized to WT d18:1/X:0 value. * P ≤ 0.05, ** P ≤ 0.01, and *** P ≤ 0.001, two-tailed Student’s t -test (SEM; n = 4 for all sections).

    Article Snippet: Chemicals for ceramide synthesis were as follows: Grubbs second-generation catalyst, N-succinimidyl palmitate, palmitoleic acid, triethylamine, and vinyl magnesium bromide (Sigma-Aldrich); ( S )- Garner aldehyde (TCI America); oct-7-enal (Novel Chemical Solutions); heptyltriphenylphosphonium bromide, sodium bis (trimethylsilyl)amide, and oxalyl chloride (Alfa Aesar); d18:1 sphingosine (Avanti Polar Lipids); tetrahydrofuran (Acros Organics); and pyridine (Mallinckrodt Chemicals).

    Techniques: Two Tailed Test

    Comparative lipidomics reveals selective upregulation of diunsaturated ceramides in DKO MEF mitochondria. A: Schematic of comparative lipidomics enabling identification of lipid changes between WT and DKO samples. B: Volcano plot of detected parent ions plotted as DKO/WT fold change versus P value (n = 3); the DKO-elevated m/z 534 metabolite and its chlorine adduct are in green. C: Structure of d18:1/16:0 ceramide. The trans ).

    Journal: Journal of Lipid Research

    Article Title: Regulation of mitochondrial ceramide distribution by members of the BCL-2 family [S]

    doi: 10.1194/jlr.M058750

    Figure Lengend Snippet: Comparative lipidomics reveals selective upregulation of diunsaturated ceramides in DKO MEF mitochondria. A: Schematic of comparative lipidomics enabling identification of lipid changes between WT and DKO samples. B: Volcano plot of detected parent ions plotted as DKO/WT fold change versus P value (n = 3); the DKO-elevated m/z 534 metabolite and its chlorine adduct are in green. C: Structure of d18:1/16:0 ceramide. The trans ).

    Article Snippet: Chemicals for ceramide synthesis were as follows: Grubbs second-generation catalyst, N-succinimidyl palmitate, palmitoleic acid, triethylamine, and vinyl magnesium bromide (Sigma-Aldrich); ( S )- Garner aldehyde (TCI America); oct-7-enal (Novel Chemical Solutions); heptyltriphenylphosphonium bromide, sodium bis (trimethylsilyl)amide, and oxalyl chloride (Alfa Aesar); d18:1 sphingosine (Avanti Polar Lipids); tetrahydrofuran (Acros Organics); and pyridine (Mallinckrodt Chemicals).

    Techniques:

    Chemical synthesis. A: Synthesis of d18:2/16:0 ceramide. B: Synthesis of d18:1/16:1 ceramide.

    Journal: Journal of Lipid Research

    Article Title: Regulation of mitochondrial ceramide distribution by members of the BCL-2 family [S]

    doi: 10.1194/jlr.M058750

    Figure Lengend Snippet: Chemical synthesis. A: Synthesis of d18:2/16:0 ceramide. B: Synthesis of d18:1/16:1 ceramide.

    Article Snippet: Chemicals for ceramide synthesis were as follows: Grubbs second-generation catalyst, N-succinimidyl palmitate, palmitoleic acid, triethylamine, and vinyl magnesium bromide (Sigma-Aldrich); ( S )- Garner aldehyde (TCI America); oct-7-enal (Novel Chemical Solutions); heptyltriphenylphosphonium bromide, sodium bis (trimethylsilyl)amide, and oxalyl chloride (Alfa Aesar); d18:1 sphingosine (Avanti Polar Lipids); tetrahydrofuran (Acros Organics); and pyridine (Mallinckrodt Chemicals).

    Techniques:

    Role of ceramide in IL-1β-induced TF expression and its modulation by WIN 55,212-2 ( A ) Effect of nSMase spiroepoxide inhibitor (selective inhibitor of nSMase; 10 μM), fumonisin B 1 (selective inhibitor of ceramide synthase; 50 μM) and ISP-1 (serine palmitoyltransferase inhibitor; 10 μM) on basal and IL-1β-induced TF protein levels. Cells were pretreated with the respective inhibitor for 1 h (fumonisin B 1 , ISP) or 1.5 h (nSMase spiroepoxide inhibitor). IL-1β or vehicle were added subsequently and the incubation was continued for 8 h. ( B ) Effect of C 2 -ceramide (10 μM), dihydro-C 2 -ceramide (10 μM) and nSMase from B. cereus (10 mU/ml) on basal and IL-1β-induced TF protein levels. Cells were incubated with the respective substance in the presence or absence of IL-1β for 8 h. ( C , D ) Impact of WIN 55,212-2 (C, 10 μM) and nSMase spiroepoxide inhibitor (D, 10 μM) on basal and IL-1β-induced cellular concentrations of C 16 -ceramide. Cells were treated with IL-1β, WIN 55,212-2 or vehicle for the times indicated (C). In case of (D) cells were pretreated with nSMase spiroepoxide inhibitor for 1.5 h. IL-1β or vehicle were added subsequently and the incubation was continued for 4 h. Percent control represents comparison with vehicle-treated cells (100%) in the absence of test substance. Values are means + SEM of n = 3 per group. * P

    Journal: Oncotarget

    Article Title: Inhibition of interleukin-1β-induced endothelial tissue factor expression by the synthetic cannabinoid WIN 55,212-2

    doi: 10.18632/oncotarget.11367

    Figure Lengend Snippet: Role of ceramide in IL-1β-induced TF expression and its modulation by WIN 55,212-2 ( A ) Effect of nSMase spiroepoxide inhibitor (selective inhibitor of nSMase; 10 μM), fumonisin B 1 (selective inhibitor of ceramide synthase; 50 μM) and ISP-1 (serine palmitoyltransferase inhibitor; 10 μM) on basal and IL-1β-induced TF protein levels. Cells were pretreated with the respective inhibitor for 1 h (fumonisin B 1 , ISP) or 1.5 h (nSMase spiroepoxide inhibitor). IL-1β or vehicle were added subsequently and the incubation was continued for 8 h. ( B ) Effect of C 2 -ceramide (10 μM), dihydro-C 2 -ceramide (10 μM) and nSMase from B. cereus (10 mU/ml) on basal and IL-1β-induced TF protein levels. Cells were incubated with the respective substance in the presence or absence of IL-1β for 8 h. ( C , D ) Impact of WIN 55,212-2 (C, 10 μM) and nSMase spiroepoxide inhibitor (D, 10 μM) on basal and IL-1β-induced cellular concentrations of C 16 -ceramide. Cells were treated with IL-1β, WIN 55,212-2 or vehicle for the times indicated (C). In case of (D) cells were pretreated with nSMase spiroepoxide inhibitor for 1.5 h. IL-1β or vehicle were added subsequently and the incubation was continued for 4 h. Percent control represents comparison with vehicle-treated cells (100%) in the absence of test substance. Values are means + SEM of n = 3 per group. * P

    Article Snippet: Materials Pure standard of endogenous ceramide (C16 ) and HPLC-grade solvents for ESI–MS and lipid extraction were purchased from Sigma-Aldrich.

    Techniques: Expressing, Incubation

    Scheme showing the proposed mechanism underlying the inhibitory effect of the synthetic cannabinoid WIN 55,212-2 on endothelial expression of tissue factor (TF) The proinflammatory cytokine interleukin (IL)-1β induces TF expression in endothelial cells by separate pathways involving ceramide formation or activation of p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinases (JNK). WIN 55,212-2 inhibits IL-1β-induced TF expression via a receptor-independent pathway resulting in a suppression of neutral sphingomyelinase (nSMase)-dependent ceramide formation as well as an interference with the activation of p38 MAPK and JNK.

    Journal: Oncotarget

    Article Title: Inhibition of interleukin-1β-induced endothelial tissue factor expression by the synthetic cannabinoid WIN 55,212-2

    doi: 10.18632/oncotarget.11367

    Figure Lengend Snippet: Scheme showing the proposed mechanism underlying the inhibitory effect of the synthetic cannabinoid WIN 55,212-2 on endothelial expression of tissue factor (TF) The proinflammatory cytokine interleukin (IL)-1β induces TF expression in endothelial cells by separate pathways involving ceramide formation or activation of p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinases (JNK). WIN 55,212-2 inhibits IL-1β-induced TF expression via a receptor-independent pathway resulting in a suppression of neutral sphingomyelinase (nSMase)-dependent ceramide formation as well as an interference with the activation of p38 MAPK and JNK.

    Article Snippet: Materials Pure standard of endogenous ceramide (C16 ) and HPLC-grade solvents for ESI–MS and lipid extraction were purchased from Sigma-Aldrich.

    Techniques: Expressing, Activation Assay

    Effects of streptozotocin (STZ), high-fat diet (HFD), and myriocin (M) on diaphragm ceramide (CER) content (mean ± SEM, n = 6 per group). ∗ Difference versus control (Ctrl); # within group difference (M+ versus M−), p

    Journal: Journal of Diabetes Research

    Article Title: Changes in the Diaphragm Lipid Content after Administration of Streptozotocin and High-Fat Diet Regime

    doi: 10.1155/2017/3437169

    Figure Lengend Snippet: Effects of streptozotocin (STZ), high-fat diet (HFD), and myriocin (M) on diaphragm ceramide (CER) content (mean ± SEM, n = 6 per group). ∗ Difference versus control (Ctrl); # within group difference (M+ versus M−), p

    Article Snippet: Ceramide class of the lipids was scraped off the plates (according to an appropriate standard, Sigma-Aldrich, St. Louis, MO) and transferred into screw tubes containing pentadecanoic acid (C15:0, Sigma-Aldrich, St. Louis, MO) as an internal standard and transmethylated (14% boron trifluoride-methanol solution).

    Techniques:

    WithaD induces N-SMase activation . (A) RT-PCR analysis of N-SMases, ceramide synthase, and A-SMase. K562 and MOLT-4 cells were treated with WithaD (1.5 and 0.5 μM respectively) for 0-120 min. RNA was extracted from total cell lysate and RT-PCR was performed. The band intensity was measured. This is one representative of three independent experiments. *indicates statistically significant difference ( P

    Journal: Molecular Cancer

    Article Title: Withanolide D induces apoptosis in leukemia by targeting the activation of neutral sphingomyelinase-ceramide cascade mediated by synergistic activation of c-Jun N-terminal kinase and p38 mitogen-activated protein kinase

    doi: 10.1186/1476-4598-9-239

    Figure Lengend Snippet: WithaD induces N-SMase activation . (A) RT-PCR analysis of N-SMases, ceramide synthase, and A-SMase. K562 and MOLT-4 cells were treated with WithaD (1.5 and 0.5 μM respectively) for 0-120 min. RNA was extracted from total cell lysate and RT-PCR was performed. The band intensity was measured. This is one representative of three independent experiments. *indicates statistically significant difference ( P

    Article Snippet: Reagents Recombinant E. coli diacylglycerol (DAG) kinase, 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT), anti-ceramide antibody, standard ceramide and sphingomyelin were from Sigma (St Louis, MO).

    Techniques: Activation Assay, Reverse Transcription Polymerase Chain Reaction

    WithaD induces ceramide production in K562 and MOLT-4 cells . (A) Cells were treated with WithaD for 0-2 hr and the intracellular ceramide content were determined using anti-ceramide antibody by flow cytometer as described in Materials and methods. Representative histogram (top) and the percentages of positive cells (bottom) are shown. Results are the mean ± S.D. in duplicate in three independent experiments. Asterisk indicates statistically significant difference (P

    Journal: Molecular Cancer

    Article Title: Withanolide D induces apoptosis in leukemia by targeting the activation of neutral sphingomyelinase-ceramide cascade mediated by synergistic activation of c-Jun N-terminal kinase and p38 mitogen-activated protein kinase

    doi: 10.1186/1476-4598-9-239

    Figure Lengend Snippet: WithaD induces ceramide production in K562 and MOLT-4 cells . (A) Cells were treated with WithaD for 0-2 hr and the intracellular ceramide content were determined using anti-ceramide antibody by flow cytometer as described in Materials and methods. Representative histogram (top) and the percentages of positive cells (bottom) are shown. Results are the mean ± S.D. in duplicate in three independent experiments. Asterisk indicates statistically significant difference (P

    Article Snippet: Reagents Recombinant E. coli diacylglycerol (DAG) kinase, 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT), anti-ceramide antibody, standard ceramide and sphingomyelin were from Sigma (St Louis, MO).

    Techniques: Flow Cytometry, Cytometry

    Effect of Fumonisin B1, (GW4869) and N-SMase silencing on ceramide production, activation of MKK group of kinases and regulation of WithaD-mediated apoptosis . (A) Effect of Fumonisin B1 (10 μM) and GW4869 (10 μM) on ceramide level induced by WithaD. Cells were pretreated with inhibitors for 1 hr; incubated further 1 hr with WithaD and ceramide levels were measured by FACS. Each column represented the mean ± S.D. in duplicate in three independent experiments. *indicates statistically significant difference ( P

    Journal: Molecular Cancer

    Article Title: Withanolide D induces apoptosis in leukemia by targeting the activation of neutral sphingomyelinase-ceramide cascade mediated by synergistic activation of c-Jun N-terminal kinase and p38 mitogen-activated protein kinase

    doi: 10.1186/1476-4598-9-239

    Figure Lengend Snippet: Effect of Fumonisin B1, (GW4869) and N-SMase silencing on ceramide production, activation of MKK group of kinases and regulation of WithaD-mediated apoptosis . (A) Effect of Fumonisin B1 (10 μM) and GW4869 (10 μM) on ceramide level induced by WithaD. Cells were pretreated with inhibitors for 1 hr; incubated further 1 hr with WithaD and ceramide levels were measured by FACS. Each column represented the mean ± S.D. in duplicate in three independent experiments. *indicates statistically significant difference ( P

    Article Snippet: Reagents Recombinant E. coli diacylglycerol (DAG) kinase, 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT), anti-ceramide antibody, standard ceramide and sphingomyelin were from Sigma (St Louis, MO).

    Techniques: Activation Assay, Incubation, FACS

    Inhibition of proliferation and migration were rescued by ERK and Akt activators respectively. (A) Following incubation with rKAL and the ERK activator ceramide C6 (10 µ M) or the Akt activator SC79 (1 µ g/ml) for 24 h, hLECs cells were harvested for immunoblotting analysis to detect phosphorylation levels of ERK and Akt. (B) Inhibition of hLEC proliferation was rescued by treatment with an ERK activator. (C) Inhibition of hLECs migration was rescued by the Akt activator. (D) Histogram representing the migrated distance of hLECs. * P

    Journal: International Journal of Oncology

    Article Title: Kallistatin exerts anti-lymphangiogenic effects by inhibiting lymphatic endothelial cell proliferation, migration and tube formation

    doi: 10.3892/ijo.2017.3972

    Figure Lengend Snippet: Inhibition of proliferation and migration were rescued by ERK and Akt activators respectively. (A) Following incubation with rKAL and the ERK activator ceramide C6 (10 µ M) or the Akt activator SC79 (1 µ g/ml) for 24 h, hLECs cells were harvested for immunoblotting analysis to detect phosphorylation levels of ERK and Akt. (B) Inhibition of hLEC proliferation was rescued by treatment with an ERK activator. (C) Inhibition of hLECs migration was rescued by the Akt activator. (D) Histogram representing the migrated distance of hLECs. * P

    Article Snippet: Ceramide C6 was purchased from Sigma-Aldrich and SC79 from Selleck Chemicals (Houston, TX, USA).

    Techniques: Inhibition, Migration, Incubation

    Proposed model for the regulatory interplay of Ox-LDL, N-SMase, ceramide and p38 MAPK during the process of VSMC calcification. Ox-LDL stimulates the activity of N-SMase, which results in increased levels of ceramide. As a result, the activated ceramide signaling stimulates p38 MAPK and triggers VSMC apoptosis, leading to VSMC calcification, which can be attenuated by GW4869 treatment.

    Journal: PLoS ONE

    Article Title: Ceramide Mediates Ox-LDL-Induced Human Vascular Smooth Muscle Cell Calcification via p38 Mitogen-Activated Protein Kinase Signaling

    doi: 10.1371/journal.pone.0082379

    Figure Lengend Snippet: Proposed model for the regulatory interplay of Ox-LDL, N-SMase, ceramide and p38 MAPK during the process of VSMC calcification. Ox-LDL stimulates the activity of N-SMase, which results in increased levels of ceramide. As a result, the activated ceramide signaling stimulates p38 MAPK and triggers VSMC apoptosis, leading to VSMC calcification, which can be attenuated by GW4869 treatment.

    Article Snippet: For determination of ceramide level, the diacylglycerol (DAG) kinase (Calbiochem, USA) assay was performed as described .

    Techniques: Activity Assay

    Effect of Ox-LDL on ceramide level and neutral sphingomyelinase (N-SMase) activity in VSMCs. Cells were incubated with 50 µg/ml Ox-LDL and harvested at indicated time points (n = 3). ( A ) Ceramide levels were determined using diacylglycerol kinase assay. ( B ) The activity of N-SMase was measured using 14 C sphingomyelin as substrate. *p

    Journal: PLoS ONE

    Article Title: Ceramide Mediates Ox-LDL-Induced Human Vascular Smooth Muscle Cell Calcification via p38 Mitogen-Activated Protein Kinase Signaling

    doi: 10.1371/journal.pone.0082379

    Figure Lengend Snippet: Effect of Ox-LDL on ceramide level and neutral sphingomyelinase (N-SMase) activity in VSMCs. Cells were incubated with 50 µg/ml Ox-LDL and harvested at indicated time points (n = 3). ( A ) Ceramide levels were determined using diacylglycerol kinase assay. ( B ) The activity of N-SMase was measured using 14 C sphingomyelin as substrate. *p

    Article Snippet: For determination of ceramide level, the diacylglycerol (DAG) kinase (Calbiochem, USA) assay was performed as described .

    Techniques: Activity Assay, Incubation, Kinase Assay

    Effect of C2-ceramide on VSMC calcification. Human VSMCs were incubated in the presence of 1 µM, 5 µM, 10 µM of C2-ceramide (C2-Cer) for 14 days (n = 3). ( A ) Alizarin red was used to visualize VSMC mineralization (bar = 200 µm). ( B ) Quantification of mineral deposition was performed. *p

    Journal: PLoS ONE

    Article Title: Ceramide Mediates Ox-LDL-Induced Human Vascular Smooth Muscle Cell Calcification via p38 Mitogen-Activated Protein Kinase Signaling

    doi: 10.1371/journal.pone.0082379

    Figure Lengend Snippet: Effect of C2-ceramide on VSMC calcification. Human VSMCs were incubated in the presence of 1 µM, 5 µM, 10 µM of C2-ceramide (C2-Cer) for 14 days (n = 3). ( A ) Alizarin red was used to visualize VSMC mineralization (bar = 200 µm). ( B ) Quantification of mineral deposition was performed. *p

    Article Snippet: For determination of ceramide level, the diacylglycerol (DAG) kinase (Calbiochem, USA) assay was performed as described .

    Techniques: Incubation

    Ox-LDL-induced VSMC calcification was enhanced by ceramide treatment. Cells were treated with 50 µg/ml Ox-LDL alone or 50 µg/ml Ox-LDL in the presence of 5 µM C2-ceramide for 7 days (n = 3). ( A ) Cells were stained with alizarin red (bar = 200 µm). ( B ) Quantification of mineral deposition was performed. ( C ) The calcium content was measured using ocresolphthalein complexone method. ( D ) ALP activity was measured by spectrophotometry. *p

    Journal: PLoS ONE

    Article Title: Ceramide Mediates Ox-LDL-Induced Human Vascular Smooth Muscle Cell Calcification via p38 Mitogen-Activated Protein Kinase Signaling

    doi: 10.1371/journal.pone.0082379

    Figure Lengend Snippet: Ox-LDL-induced VSMC calcification was enhanced by ceramide treatment. Cells were treated with 50 µg/ml Ox-LDL alone or 50 µg/ml Ox-LDL in the presence of 5 µM C2-ceramide for 7 days (n = 3). ( A ) Cells were stained with alizarin red (bar = 200 µm). ( B ) Quantification of mineral deposition was performed. ( C ) The calcium content was measured using ocresolphthalein complexone method. ( D ) ALP activity was measured by spectrophotometry. *p

    Article Snippet: For determination of ceramide level, the diacylglycerol (DAG) kinase (Calbiochem, USA) assay was performed as described .

    Techniques: Staining, ALP Assay, Activity Assay, Spectrophotometry

    Effect of GW4869 on Ox-LDL-induced apoptosis in cultured VSMCs (n = 3). Cells were incubated in control medium (in DMSO), 50 µg/ml Ox-LDL alone (in DMSO), 50 µg/ml Ox-LDL in the presence of 20 µM GW4869 (in DMSO) or 50 µg/ml Ox-LDL in the presence of 20 µM GW4869 and 5 µM C2-ceramide (in DMSO) for 24 hours. ( A ) The percentage of apoptotic cells was then assessed. ( B ) Caspase-3 activity was measured and results are shown as percentage of control. ( C ) p-Bcl2 and p-Bad protein levels were detected by immunoblot analysis. ( D ) p-Bcl2 and p-Bad protein levels were quantified and normalized to total Bcl2 and Bad levels. ( E ) Cells were treated with/without 20 µM GW4869 and 5 µM C2-ceramide in the presence of Ox-LDL for 7 days. The calcium content was quantified using ocresolphthalein complexone method. *p

    Journal: PLoS ONE

    Article Title: Ceramide Mediates Ox-LDL-Induced Human Vascular Smooth Muscle Cell Calcification via p38 Mitogen-Activated Protein Kinase Signaling

    doi: 10.1371/journal.pone.0082379

    Figure Lengend Snippet: Effect of GW4869 on Ox-LDL-induced apoptosis in cultured VSMCs (n = 3). Cells were incubated in control medium (in DMSO), 50 µg/ml Ox-LDL alone (in DMSO), 50 µg/ml Ox-LDL in the presence of 20 µM GW4869 (in DMSO) or 50 µg/ml Ox-LDL in the presence of 20 µM GW4869 and 5 µM C2-ceramide (in DMSO) for 24 hours. ( A ) The percentage of apoptotic cells was then assessed. ( B ) Caspase-3 activity was measured and results are shown as percentage of control. ( C ) p-Bcl2 and p-Bad protein levels were detected by immunoblot analysis. ( D ) p-Bcl2 and p-Bad protein levels were quantified and normalized to total Bcl2 and Bad levels. ( E ) Cells were treated with/without 20 µM GW4869 and 5 µM C2-ceramide in the presence of Ox-LDL for 7 days. The calcium content was quantified using ocresolphthalein complexone method. *p

    Article Snippet: For determination of ceramide level, the diacylglycerol (DAG) kinase (Calbiochem, USA) assay was performed as described .

    Techniques: Cell Culture, Incubation, Activity Assay

    Effect SB203580 of on Ox-LDL-induced mineralization in cultured VSMCs (n = 3). Cells were grown in control medium (in DMSO), 50 µg/ml Ox-LDL alone (in DMSO), 50 µg/ml Ox-LDL supplemented with 5 µM SB203580 (in DMSO), 5 µM C2-ceramide alone (in DMSO) or 5 µM C2-ceramide supplemented with 5 µM SB203580 (in DMSO) for 24 hours. ( A ) p-p38 MAPK protein levels were detected by immunoblot analysis. ( B ) The percentage of apoptotic cells was then assessed. ( C ) Caspase-3 activity was measured in a parallel experiment. Cells were treated with SB203580 in the presence of Ox-LDL for 7 days. ( D ) The calcium content was measured using ocresolphthalein complexone method. ( E ) Msx2 mRNA expression was determined by quantitative real-time PCR after cells were treated with/without SB203580. *p

    Journal: PLoS ONE

    Article Title: Ceramide Mediates Ox-LDL-Induced Human Vascular Smooth Muscle Cell Calcification via p38 Mitogen-Activated Protein Kinase Signaling

    doi: 10.1371/journal.pone.0082379

    Figure Lengend Snippet: Effect SB203580 of on Ox-LDL-induced mineralization in cultured VSMCs (n = 3). Cells were grown in control medium (in DMSO), 50 µg/ml Ox-LDL alone (in DMSO), 50 µg/ml Ox-LDL supplemented with 5 µM SB203580 (in DMSO), 5 µM C2-ceramide alone (in DMSO) or 5 µM C2-ceramide supplemented with 5 µM SB203580 (in DMSO) for 24 hours. ( A ) p-p38 MAPK protein levels were detected by immunoblot analysis. ( B ) The percentage of apoptotic cells was then assessed. ( C ) Caspase-3 activity was measured in a parallel experiment. Cells were treated with SB203580 in the presence of Ox-LDL for 7 days. ( D ) The calcium content was measured using ocresolphthalein complexone method. ( E ) Msx2 mRNA expression was determined by quantitative real-time PCR after cells were treated with/without SB203580. *p

    Article Snippet: For determination of ceramide level, the diacylglycerol (DAG) kinase (Calbiochem, USA) assay was performed as described .

    Techniques: Cell Culture, Activity Assay, Expressing, Real-time Polymerase Chain Reaction

    Cell death-modulating activities of ceramides. ( A ) Nuclear morphology of wild-type protoplasts after control or C2 ceramide treatments. Protoplasts were stained for nuclei using Hoechst33342 16 h after treatment with 30 µM C2 ceramide. Protoplasts showed 32% viability. Bar, 10 µm. The percentage of protoplasts of each genotype with the morphologies shown is indicated below each picture. ( B ) Ceramide-induced cell death in wild-type and acd5 protoplasts. Protoplasts were treated with C2 ceramide (open symbols) or C2 dihydroceramide (closed symbols) for 10 h. (Circles) Wild-type protoplasts; (triangles) acd5 protoplasts. Standard deviations are shown ( n = 3). Some symbols obscure the error bars. ( C ) Cell death-blocking effects of C2 ceramide-1-phosphate (C2-1P). Protoplasts were left untreated or were treated with 0.2% ethanol (solvent in which ceramides were dissolved), 30 µM C2 ceramide, 30 µM C2 ceramide + 10 µM C2 ceramide-1-phosphate, or 10 µM C2 ceramide-1-phosphate for 18 h. Standard deviations are shown ( n = 3). Letters indicate that values are different using Student's t -test ( P

    Journal: Genes & Development

    Article Title: Ceramides modulate programmed cell death in plants

    doi: 10.1101/gad.1140503

    Figure Lengend Snippet: Cell death-modulating activities of ceramides. ( A ) Nuclear morphology of wild-type protoplasts after control or C2 ceramide treatments. Protoplasts were stained for nuclei using Hoechst33342 16 h after treatment with 30 µM C2 ceramide. Protoplasts showed 32% viability. Bar, 10 µm. The percentage of protoplasts of each genotype with the morphologies shown is indicated below each picture. ( B ) Ceramide-induced cell death in wild-type and acd5 protoplasts. Protoplasts were treated with C2 ceramide (open symbols) or C2 dihydroceramide (closed symbols) for 10 h. (Circles) Wild-type protoplasts; (triangles) acd5 protoplasts. Standard deviations are shown ( n = 3). Some symbols obscure the error bars. ( C ) Cell death-blocking effects of C2 ceramide-1-phosphate (C2-1P). Protoplasts were left untreated or were treated with 0.2% ethanol (solvent in which ceramides were dissolved), 30 µM C2 ceramide, 30 µM C2 ceramide + 10 µM C2 ceramide-1-phosphate, or 10 µM C2 ceramide-1-phosphate for 18 h. Standard deviations are shown ( n = 3). Letters indicate that values are different using Student's t -test ( P

    Article Snippet: Natural ceramide and sphingosine were purchased from Sigma.

    Techniques: Staining, Blocking Assay

    CERK activity of ACD5. ( A ) Autoradiographs of TLC plates detecting 32 P incorporation from [γ- 32 P]ATP (3000 Ci/mmole) into lipid substrates. (rACD5) Recombinant ACD5; (DAGK) diacylglycerol kinase. ( B ) Quantitative analysis of relative ACD5 CERK activity. Standard deviations are indicated ( n = 3). ACD5 activity was 344 and 36.1 fmole/min/pmole of enzyme, using C8 and natural ceramide (cer), respectively. ( C ) Kinetic analysis of recombinant ACD5. Standard deviations are indicated ( n = 3). ( D ) Effect of various cations (100 mM) on recombinant ACD5 enzyme activity. ( E ) Conversion of C8 ceramide to C8 ceramide-1-phospate by E. coli DAGK and crude extracts from 33-day-old plants; 2.5-fold more acd5 protein extract than wild-type ( ACD5 + ) extract was used in the assay. ( F ) Accumulation of ACD5 CERK substrates in acd5 and wild-type lipid extracts. Recombinant ACD5 or vector control protein (-) was used to phosphorylate ceramide-enriched acd5 and wild-type lipid extracts, and the phosphorylated products were run on TLC. Darker bands in acd5 lanes indicate that more CERK substrate was present in the acd5 lipid extracts than in the wild-type lipid extracts; 1.5-fold more lipids were loaded on the TLC plate from the 25-day-old vs. 35-day-old material.

    Journal: Genes & Development

    Article Title: Ceramides modulate programmed cell death in plants

    doi: 10.1101/gad.1140503

    Figure Lengend Snippet: CERK activity of ACD5. ( A ) Autoradiographs of TLC plates detecting 32 P incorporation from [γ- 32 P]ATP (3000 Ci/mmole) into lipid substrates. (rACD5) Recombinant ACD5; (DAGK) diacylglycerol kinase. ( B ) Quantitative analysis of relative ACD5 CERK activity. Standard deviations are indicated ( n = 3). ACD5 activity was 344 and 36.1 fmole/min/pmole of enzyme, using C8 and natural ceramide (cer), respectively. ( C ) Kinetic analysis of recombinant ACD5. Standard deviations are indicated ( n = 3). ( D ) Effect of various cations (100 mM) on recombinant ACD5 enzyme activity. ( E ) Conversion of C8 ceramide to C8 ceramide-1-phospate by E. coli DAGK and crude extracts from 33-day-old plants; 2.5-fold more acd5 protein extract than wild-type ( ACD5 + ) extract was used in the assay. ( F ) Accumulation of ACD5 CERK substrates in acd5 and wild-type lipid extracts. Recombinant ACD5 or vector control protein (-) was used to phosphorylate ceramide-enriched acd5 and wild-type lipid extracts, and the phosphorylated products were run on TLC. Darker bands in acd5 lanes indicate that more CERK substrate was present in the acd5 lipid extracts than in the wild-type lipid extracts; 1.5-fold more lipids were loaded on the TLC plate from the 25-day-old vs. 35-day-old material.

    Article Snippet: Natural ceramide and sphingosine were purchased from Sigma.

    Techniques: Activity Assay, Thin Layer Chromatography, Recombinant, Plasmid Preparation

    C 24:1 ceramide restores NP rosettes in ceramide-depleted cells. Human ES cells were differentiated for 5 d in the presence of 20 μM FB1 with or without 2 μM C 24:1 ceramide, followed by immunocytochemistry for acetylated tubulin (mouse or rabbit IgG), aPKC (rabbit IgG), ceramide (mouse IgM), or nestin (mouse IgG). A. Formation of neural rosettes (arrows) is severely reduced in ceramide-depleted NPs. (B) Treatment with C 24:1 ceramide restores the number of NP rosettes ( > 100 μm diameter) similar to the effect of aPKC inhibition with Go6983. N = 3, p

    Journal: Molecular Biology of the Cell

    Article Title: Primary cilia in stem cells and neural progenitors are regulated by neutral sphingomyelinase 2 and ceramide

    doi: 10.1091/mbc.E13-12-0730

    Figure Lengend Snippet: C 24:1 ceramide restores NP rosettes in ceramide-depleted cells. Human ES cells were differentiated for 5 d in the presence of 20 μM FB1 with or without 2 μM C 24:1 ceramide, followed by immunocytochemistry for acetylated tubulin (mouse or rabbit IgG), aPKC (rabbit IgG), ceramide (mouse IgM), or nestin (mouse IgG). A. Formation of neural rosettes (arrows) is severely reduced in ceramide-depleted NPs. (B) Treatment with C 24:1 ceramide restores the number of NP rosettes ( > 100 μm diameter) similar to the effect of aPKC inhibition with Go6983. N = 3, p

    Article Snippet: Preparation and characterization of high-affinity C24 and C24:1 ceramide antibodies A C24 ceramide emulsion was prepared with keyhole limpet hemocyanin in phosphate-buffered saline (PBS) and Freund's complete adjuvant (Sigma-Aldrich) as described previously ( ).

    Techniques: Immunocytochemistry, Inhibition

    C 16:0 ceramide is localized at primary cilia, whereas C 24:0/24:1 ceramide is at primary cilia and the apicolateral cell membrane. Neural differentiation was performed in the presence or absence of FB1 or GW4869, followed by immunocytochemistry using antibodies against C 16:0 ceramide (anti-cerIgM) or C 24:0/C24:1 ceramide (anti-C24). DAPI staining is pseudocolored in gray, anti-ceramide IgM in red, anti-C24 ceramide IgG in green, and acetylated tubulin in blue. (A) Control, apical plane (arrow points at colocalization of anti-cerIgM, anti-C24, and anti-acetylated tubulin antibodies at primary cilia). (B) Control, apicolateral plane (arrow points at apicolateral cell membrane). (C) FB1-treated cells. (D) GW4869-treated cells. Bars, 10 μm.

    Journal: Molecular Biology of the Cell

    Article Title: Primary cilia in stem cells and neural progenitors are regulated by neutral sphingomyelinase 2 and ceramide

    doi: 10.1091/mbc.E13-12-0730

    Figure Lengend Snippet: C 16:0 ceramide is localized at primary cilia, whereas C 24:0/24:1 ceramide is at primary cilia and the apicolateral cell membrane. Neural differentiation was performed in the presence or absence of FB1 or GW4869, followed by immunocytochemistry using antibodies against C 16:0 ceramide (anti-cerIgM) or C 24:0/C24:1 ceramide (anti-C24). DAPI staining is pseudocolored in gray, anti-ceramide IgM in red, anti-C24 ceramide IgG in green, and acetylated tubulin in blue. (A) Control, apical plane (arrow points at colocalization of anti-cerIgM, anti-C24, and anti-acetylated tubulin antibodies at primary cilia). (B) Control, apicolateral plane (arrow points at apicolateral cell membrane). (C) FB1-treated cells. (D) GW4869-treated cells. Bars, 10 μm.

    Article Snippet: Preparation and characterization of high-affinity C24 and C24:1 ceramide antibodies A C24 ceramide emulsion was prepared with keyhole limpet hemocyanin in phosphate-buffered saline (PBS) and Freund's complete adjuvant (Sigma-Aldrich) as described previously ( ).

    Techniques: Immunocytochemistry, Staining

    Ceramide is colocalized with acetylated tubulin in primary cilia and the mitotic spindle. Human ES cells were cultivated feeder free and then subjected to immunocytochemistry using antibodies against ceramide (rabbit IgG or mouse IgM, red) and acetylated tubulin (green). (A) Interphase; arrow points at primary cilium. Bar, 10 μm. (B) Metaphase. Bar, 5 μm. (C, D) Neural differentiation suppresses expression of Oct-4 concurrent with elevated expression of ceramide and formation of primary cilia (C is before and D is after neural differentiation). Bar, 20 μm. (E) Primary cilia are formed in nestin-expressing NPs in neural rosettes. In NPs, ceramide is colocalized with primary cilia and vesicles at the cilium bases (arrows). Bar, 5 μm. (F) A z -scan of individual cell in neural rosette. Arrow indicates colocalization of ceramide with acetylated tubulin in primary cilium. Right, apical plane of area used for z -scan.

    Journal: Molecular Biology of the Cell

    Article Title: Primary cilia in stem cells and neural progenitors are regulated by neutral sphingomyelinase 2 and ceramide

    doi: 10.1091/mbc.E13-12-0730

    Figure Lengend Snippet: Ceramide is colocalized with acetylated tubulin in primary cilia and the mitotic spindle. Human ES cells were cultivated feeder free and then subjected to immunocytochemistry using antibodies against ceramide (rabbit IgG or mouse IgM, red) and acetylated tubulin (green). (A) Interphase; arrow points at primary cilium. Bar, 10 μm. (B) Metaphase. Bar, 5 μm. (C, D) Neural differentiation suppresses expression of Oct-4 concurrent with elevated expression of ceramide and formation of primary cilia (C is before and D is after neural differentiation). Bar, 20 μm. (E) Primary cilia are formed in nestin-expressing NPs in neural rosettes. In NPs, ceramide is colocalized with primary cilia and vesicles at the cilium bases (arrows). Bar, 5 μm. (F) A z -scan of individual cell in neural rosette. Arrow indicates colocalization of ceramide with acetylated tubulin in primary cilium. Right, apical plane of area used for z -scan.

    Article Snippet: Preparation and characterization of high-affinity C24 and C24:1 ceramide antibodies A C24 ceramide emulsion was prepared with keyhole limpet hemocyanin in phosphate-buffered saline (PBS) and Freund's complete adjuvant (Sigma-Aldrich) as described previously ( ).

    Techniques: Immunocytochemistry, Expressing

    Ceramide depletion leads to translocation of aPKC from the primary cilium and the apicolateral cell membrane to the cytosol. NPs were analyzed using immunocytochemistry with anti-ceramide mouse IgM (red) and antibodies against acetylated tubulin (mouse IgG, green) and aPKC (C20, rabbit IgG, blue). (A, B) Mitotic cells. Note colocalization of ceramide with mitotic spindle. Bar, 5 μm. (C) Interphase (arrow points at primary cilium). 4′,6-Diamidino-2-phenylindole (DAPI) staining is pseudocolored in gray. Bar, 2 μm. (D–F) NPs differentiated from control (D) and FB1- (E) and GW4869-treated (F) human ES cells, followed by immunocytochemistry using antibodies as described for (A–C). aPKC is colocalized with the primary cilium (arrow) and the apicolateral cell membrane (arrowhead) of control cells (D). Colocalization is lost when cells are depleted of ceramide by treatment with FB1 (E) or GW4869 (F). Bars, 10 μm.

    Journal: Molecular Biology of the Cell

    Article Title: Primary cilia in stem cells and neural progenitors are regulated by neutral sphingomyelinase 2 and ceramide

    doi: 10.1091/mbc.E13-12-0730

    Figure Lengend Snippet: Ceramide depletion leads to translocation of aPKC from the primary cilium and the apicolateral cell membrane to the cytosol. NPs were analyzed using immunocytochemistry with anti-ceramide mouse IgM (red) and antibodies against acetylated tubulin (mouse IgG, green) and aPKC (C20, rabbit IgG, blue). (A, B) Mitotic cells. Note colocalization of ceramide with mitotic spindle. Bar, 5 μm. (C) Interphase (arrow points at primary cilium). 4′,6-Diamidino-2-phenylindole (DAPI) staining is pseudocolored in gray. Bar, 2 μm. (D–F) NPs differentiated from control (D) and FB1- (E) and GW4869-treated (F) human ES cells, followed by immunocytochemistry using antibodies as described for (A–C). aPKC is colocalized with the primary cilium (arrow) and the apicolateral cell membrane (arrowhead) of control cells (D). Colocalization is lost when cells are depleted of ceramide by treatment with FB1 (E) or GW4869 (F). Bars, 10 μm.

    Article Snippet: Preparation and characterization of high-affinity C24 and C24:1 ceramide antibodies A C24 ceramide emulsion was prepared with keyhole limpet hemocyanin in phosphate-buffered saline (PBS) and Freund's complete adjuvant (Sigma-Aldrich) as described previously ( ).

    Techniques: Translocation Assay, Immunocytochemistry, Staining

    In ceramide-depleted cells, activation of cytosolic aPKC reduces ciliogenesis, whereas inhibition of HDAC6 and AurA rescues cilia. (A) Control and FB1- or GW4869-treated NPs were subjected to subcellular fractionation using differential centrifugation. The cytosolic fraction of ceramide-depleted cells contains a phosphorylated, 55-kDa isoform of aPKC (aPKCcat). In addition, there are 65- and 45-kDa isoforms, the latter of which is phosphorylated but it is also present in control cells. (B) Inhibition of aPKC with pseudo substrate inhibitor peptide of aPKC (PZI) or Go6983 (Go), or AurA and HDAC6 inhibition with PHA-680632 (PHA) and tubacin (Tuba), can partially rescue ciliogenesis in FB1-treated NPs. N ≥ 5, * p

    Journal: Molecular Biology of the Cell

    Article Title: Primary cilia in stem cells and neural progenitors are regulated by neutral sphingomyelinase 2 and ceramide

    doi: 10.1091/mbc.E13-12-0730

    Figure Lengend Snippet: In ceramide-depleted cells, activation of cytosolic aPKC reduces ciliogenesis, whereas inhibition of HDAC6 and AurA rescues cilia. (A) Control and FB1- or GW4869-treated NPs were subjected to subcellular fractionation using differential centrifugation. The cytosolic fraction of ceramide-depleted cells contains a phosphorylated, 55-kDa isoform of aPKC (aPKCcat). In addition, there are 65- and 45-kDa isoforms, the latter of which is phosphorylated but it is also present in control cells. (B) Inhibition of aPKC with pseudo substrate inhibitor peptide of aPKC (PZI) or Go6983 (Go), or AurA and HDAC6 inhibition with PHA-680632 (PHA) and tubacin (Tuba), can partially rescue ciliogenesis in FB1-treated NPs. N ≥ 5, * p

    Article Snippet: Preparation and characterization of high-affinity C24 and C24:1 ceramide antibodies A C24 ceramide emulsion was prepared with keyhole limpet hemocyanin in phosphate-buffered saline (PBS) and Freund's complete adjuvant (Sigma-Aldrich) as described previously ( ).

    Techniques: Activation Assay, Inhibition, Fractionation, Centrifugation

    Ceramide depletion reduces ciliogenesis, whereas addition of ceramide rescues cilium formation. Mass spectrometric analysis of ceramide species in undifferentiated human ES cells and ES cell-derived NPs (A) and HPTLC analysis (B) of ceramide in NPs in the presence or absence of inhibitors for ceramide generation (FB1 or GW4869). Arrow in B points at ceramide band stained with cupric acetate/charring. (C) Proportion of ciliated cells (length of cilia > 1 μm) in NPs with reduced ceramide levels and after the addition of exogenous ceramide (C16, C 16:0 ceramide; C18, C 18:0 ceramide; C 24:1 , C 24:1 ceramide) or ceramide analogues (S16, N -palmitoyl serinol). N > 5; p value for difference between controls and FB1- or GW4869-treated cells, ** p

    Journal: Molecular Biology of the Cell

    Article Title: Primary cilia in stem cells and neural progenitors are regulated by neutral sphingomyelinase 2 and ceramide

    doi: 10.1091/mbc.E13-12-0730

    Figure Lengend Snippet: Ceramide depletion reduces ciliogenesis, whereas addition of ceramide rescues cilium formation. Mass spectrometric analysis of ceramide species in undifferentiated human ES cells and ES cell-derived NPs (A) and HPTLC analysis (B) of ceramide in NPs in the presence or absence of inhibitors for ceramide generation (FB1 or GW4869). Arrow in B points at ceramide band stained with cupric acetate/charring. (C) Proportion of ciliated cells (length of cilia > 1 μm) in NPs with reduced ceramide levels and after the addition of exogenous ceramide (C16, C 16:0 ceramide; C18, C 18:0 ceramide; C 24:1 , C 24:1 ceramide) or ceramide analogues (S16, N -palmitoyl serinol). N > 5; p value for difference between controls and FB1- or GW4869-treated cells, ** p

    Article Snippet: Preparation and characterization of high-affinity C24 and C24:1 ceramide antibodies A C24 ceramide emulsion was prepared with keyhole limpet hemocyanin in phosphate-buffered saline (PBS) and Freund's complete adjuvant (Sigma-Aldrich) as described previously ( ).

    Techniques: Derivative Assay, High Performance Thin Layer Chromatography, Staining

    C 24:1 ceramide promotes neural process formation in human ES and iPS cells. Human ES or iPS cells were treated as described in the legend to Figure 6 . (A) Colocalization of acetylated tubulin (green) and Map-2 (red) shows that many processes originate in neural progenitors or differentiated neurons. (B–D) Acetylated tubulin–labeled processes and nestin-labeled NPs are also visible in iPS cells that were differentiated following the protocol used for human ES cells. Neural processes of C 24:1 ceramide–treated iPS cells are elongated ( > 200 μm) and colabeled for Map-2. Bars, 20 μm (A, B, D), 200 μm (C).

    Journal: Molecular Biology of the Cell

    Article Title: Primary cilia in stem cells and neural progenitors are regulated by neutral sphingomyelinase 2 and ceramide

    doi: 10.1091/mbc.E13-12-0730

    Figure Lengend Snippet: C 24:1 ceramide promotes neural process formation in human ES and iPS cells. Human ES or iPS cells were treated as described in the legend to Figure 6 . (A) Colocalization of acetylated tubulin (green) and Map-2 (red) shows that many processes originate in neural progenitors or differentiated neurons. (B–D) Acetylated tubulin–labeled processes and nestin-labeled NPs are also visible in iPS cells that were differentiated following the protocol used for human ES cells. Neural processes of C 24:1 ceramide–treated iPS cells are elongated ( > 200 μm) and colabeled for Map-2. Bars, 20 μm (A, B, D), 200 μm (C).

    Article Snippet: Preparation and characterization of high-affinity C24 and C24:1 ceramide antibodies A C24 ceramide emulsion was prepared with keyhole limpet hemocyanin in phosphate-buffered saline (PBS) and Freund's complete adjuvant (Sigma-Aldrich) as described previously ( ).

    Techniques: Labeling

    Working model for ER–Golgi contact-mediated regulation of transport carrier biogenesis. Ceramide (Cer) and cholesterol (Chol) are transported from the ER to the trans- Golgi/TGN by VAP-CERT and VAP-OSBP-Sac1 complexes, respectively, at the membrane contact site. CARTS biogenesis is controlled in two ways: 1) DAG-dependent recruitment of PKD and 2) cholesterol- and SM-rich microdomain organization. PI4P transported from the trans- Golgi/TGN to the ER is hydrolyzed by Sac1, which is recruited to a VAP-OSBP complex formed at a specialized ER subdomain closely apposed to the trans- Golgi/TGN. Reagents used in this study are also shown: 25-OH treatment inhibits both cholesterol synthesis and transfer; d -ceramide-C6 ( d -Cer-C6) treatment disrupts cholesterol- and SM-rich microdomain organization.

    Journal: Molecular Biology of the Cell

    Article Title: CARTS biogenesis requires VAP–lipid transfer protein complexes functioning at the endoplasmic reticulum–Golgi interface

    doi: 10.1091/mbc.E15-08-0599

    Figure Lengend Snippet: Working model for ER–Golgi contact-mediated regulation of transport carrier biogenesis. Ceramide (Cer) and cholesterol (Chol) are transported from the ER to the trans- Golgi/TGN by VAP-CERT and VAP-OSBP-Sac1 complexes, respectively, at the membrane contact site. CARTS biogenesis is controlled in two ways: 1) DAG-dependent recruitment of PKD and 2) cholesterol- and SM-rich microdomain organization. PI4P transported from the trans- Golgi/TGN to the ER is hydrolyzed by Sac1, which is recruited to a VAP-OSBP complex formed at a specialized ER subdomain closely apposed to the trans- Golgi/TGN. Reagents used in this study are also shown: 25-OH treatment inhibits both cholesterol synthesis and transfer; d -ceramide-C6 ( d -Cer-C6) treatment disrupts cholesterol- and SM-rich microdomain organization.

    Article Snippet: Nocodazole, d -ceramide-C6 (N -hexanoyl-d -spingosin), and 25-OH were purchased from Sigma-Aldrich.

    Techniques:

    Sphingosine induces IL-1 release from LPS-primed macrophages. (A, B) LPS-treated (1 mg/mL, 2h) murine peritoneal macrophages were incubated for 1 h with the indicated concentration of (A) sphingosine (Sph) or (B) the Sph analogue FTY720. IL-1β in the supernatants was quantified by ELISA (top). Processing of pro-IL-1β (band at 31 to 17 kDa) was determined by western blot (bottom). (C) The structures of key mediators in the sphingolipid pathway are shown, and the structures of the Sph analogue FTY720, the S1P analogue FTY720-P and the sphingosine kinase inhibitor DMS are provided for comparison. (D, E) LPS-treated peritoneal macrophages were also incubated with the indicated concentrations of (D) ceramide or S1P or (E) 20 μM Sph, DMS, FTY720 and FTY720-P (FTYP) for 1h. IL-1β was quantified in supernatants by ELISA and pro-IL-1β processing to mature IL-1β was determined by western blot (E bottom). Data are shown as the mean 7 1 SD of three experiments. ***p

    Journal: European Journal of Immunology

    Article Title: Sphingosine regulates the NLRP3-inflammasome and IL-1 β release from macrophages

    doi: 10.1002/eji.201142079

    Figure Lengend Snippet: Sphingosine induces IL-1 release from LPS-primed macrophages. (A, B) LPS-treated (1 mg/mL, 2h) murine peritoneal macrophages were incubated for 1 h with the indicated concentration of (A) sphingosine (Sph) or (B) the Sph analogue FTY720. IL-1β in the supernatants was quantified by ELISA (top). Processing of pro-IL-1β (band at 31 to 17 kDa) was determined by western blot (bottom). (C) The structures of key mediators in the sphingolipid pathway are shown, and the structures of the Sph analogue FTY720, the S1P analogue FTY720-P and the sphingosine kinase inhibitor DMS are provided for comparison. (D, E) LPS-treated peritoneal macrophages were also incubated with the indicated concentrations of (D) ceramide or S1P or (E) 20 μM Sph, DMS, FTY720 and FTY720-P (FTYP) for 1h. IL-1β was quantified in supernatants by ELISA and pro-IL-1β processing to mature IL-1β was determined by western blot (E bottom). Data are shown as the mean 7 1 SD of three experiments. ***p

    Article Snippet: Ac-YVAD-CHO, E-64, DMS, Ca074-Me and Z-Phe-Arg-AMC were from Merck Chemicals (2R,4R)-4-aminopyrrolidine (APDC), ceramide and A740003 were from Tocris Bioscience.

    Techniques: Incubation, Concentration Assay, Enzyme-linked Immunosorbent Assay, Western Blot

    Effect of ceramide on insulin-induced phosphorylation of PKB/Akt in control human fibroblast versus caveolin-1–overexpressing human fibroblast. A : Control human fibroblast and caveolin-1–overexpressing human fibroblast lysates were immunoblotted with antibodies against either PKCζ, PP2A, native PKB/Akt, GFP, or caveolin-1. B : Human fibroblasts were treated with 500 μmol/l OKA for 30 min and 100 nmol/l insulin for the last 10 min before being lysed. Cell lysates were immunoblotted with antibodies against either native PKB/Akt or Ser473 PKB/Akt. C : Control human fibroblast and caveolin-1–overexpressing human fibroblast were preincubated with 100 μmol/l C2-ceramide for 2 h, followed by 500 μmol/l OKA for the last 30 min. Then, 100 nmol/l insulin was added to the cells for 10 min before being lysed. Cell lysates were immunoblotted with antibodies against native PKB/Akt, Ser473 PKB/Akt, Ser21/9 GSK3α/β, PKCζ, and GFP. Scanning densitometry was performed to quantify changes in Ser473 PKB/Akt abundance in cell lysates. Bars represent mean ± SEM. *Significant change, P

    Journal: Diabetes

    Article Title: Plasma Membrane Subdomain Compartmentalization Contributes to Distinct Mechanisms of Ceramide Action on Insulin Signaling

    doi: 10.2337/db09-0897

    Figure Lengend Snippet: Effect of ceramide on insulin-induced phosphorylation of PKB/Akt in control human fibroblast versus caveolin-1–overexpressing human fibroblast. A : Control human fibroblast and caveolin-1–overexpressing human fibroblast lysates were immunoblotted with antibodies against either PKCζ, PP2A, native PKB/Akt, GFP, or caveolin-1. B : Human fibroblasts were treated with 500 μmol/l OKA for 30 min and 100 nmol/l insulin for the last 10 min before being lysed. Cell lysates were immunoblotted with antibodies against either native PKB/Akt or Ser473 PKB/Akt. C : Control human fibroblast and caveolin-1–overexpressing human fibroblast were preincubated with 100 μmol/l C2-ceramide for 2 h, followed by 500 μmol/l OKA for the last 30 min. Then, 100 nmol/l insulin was added to the cells for 10 min before being lysed. Cell lysates were immunoblotted with antibodies against native PKB/Akt, Ser473 PKB/Akt, Ser21/9 GSK3α/β, PKCζ, and GFP. Scanning densitometry was performed to quantify changes in Ser473 PKB/Akt abundance in cell lysates. Bars represent mean ± SEM. *Significant change, P

    Article Snippet: C2 -ceramide was obtained from Tocris and OKA from Calbiochem.

    Techniques:

    Effects of high tau protein concentration on targeted migration of NSCs in the injured spinal cord of rats (Transwell assay). (A) Cresyl violet staining of NSCs in the control group and at different tau concentrations (0, 0.1, 0.5, 1.0 mg/mL) in the lower chamber (light microscope, × 100). (B) Cresyl violet staining of NSCs in the control, OA, C2-ceramide and unpretreated NSC groups (light microscope, × 100). (C) Optical density of cresyl violet staining of NSCs in the control group and at different tau concentrations (0, 0.1, 0.5, 1.0 mg/mL) in the lower chamber (no significant differences observed between tau concentrations). (D) Optical density of cresyl violet staining of NSCs in the control group, OA group, C2-ceramide group and unpretreated NSCs group (optical density was significantly lower in the OA and C2-ceramide groups than in the unpretreated NSCs group). * P

    Journal: Neural Regeneration Research

    Article Title: Effects of microtubule-associated protein tau expression on neural stem cell migration after spinal cord injury

    doi: 10.4103/1673-5374.177744

    Figure Lengend Snippet: Effects of high tau protein concentration on targeted migration of NSCs in the injured spinal cord of rats (Transwell assay). (A) Cresyl violet staining of NSCs in the control group and at different tau concentrations (0, 0.1, 0.5, 1.0 mg/mL) in the lower chamber (light microscope, × 100). (B) Cresyl violet staining of NSCs in the control, OA, C2-ceramide and unpretreated NSC groups (light microscope, × 100). (C) Optical density of cresyl violet staining of NSCs in the control group and at different tau concentrations (0, 0.1, 0.5, 1.0 mg/mL) in the lower chamber (no significant differences observed between tau concentrations). (D) Optical density of cresyl violet staining of NSCs in the control group, OA group, C2-ceramide group and unpretreated NSCs group (optical density was significantly lower in the OA and C2-ceramide groups than in the unpretreated NSCs group). * P

    Article Snippet: NSCs pretreated with okadaic acid (OA) (10 nM; Sigma-Aldrich), NSCs pretreated with C2-ceramide (1 μM; Sigma-Aldrich) and unpretreated NSCs were added to the corresponding upper chamber, respectively.

    Techniques: Protein Concentration, Migration, Transwell Assay, Staining, Light Microscopy

    CERT knockdown does not lead to a decrease in HexCer levels. A: Total sphingomyelin and HexCer levels of Hela cells treated with siRNA against the ceramide transport protein CERT. Scrambled (SCR) siRNA was used as control. B: Individual sphingomyelin

    Journal: Journal of Lipid Research

    Article Title: Glycosylphosphatidylinositol anchors regulate glycosphingolipid levels [S]

    doi: 10.1194/jlr.M025692

    Figure Lengend Snippet: CERT knockdown does not lead to a decrease in HexCer levels. A: Total sphingomyelin and HexCer levels of Hela cells treated with siRNA against the ceramide transport protein CERT. Scrambled (SCR) siRNA was used as control. B: Individual sphingomyelin

    Article Snippet: DLPC 12:0/12:0 (850335), PE 17:0/14:1 (PE31:1, LM-1104), PI 17:0/14:1 (PI31:1, LM-1504), PS 17:0/14:1 (PS31:1, LM-1304), C17:0 ceramide (860517), C12:0 SM (860583) and Glucosyl C8:0 Cer (860540) were used as internal lipid standards and were purchased from Avanti Polar Lipids Inc. (Alabaster, AL).

    Techniques:

    Ceramide profile of GPI anchor-deficient cells. A: Total ceramide (Cer) levels in F21 wild-type and GPI anchor mutants DPM3, PIG-X, PIG-F, and PGAP2. B: Total Cer levels in C311 wild-type and GPI anchor mutant cell lines PIG-O and PIG-U. C: Individual

    Journal: Journal of Lipid Research

    Article Title: Glycosylphosphatidylinositol anchors regulate glycosphingolipid levels [S]

    doi: 10.1194/jlr.M025692

    Figure Lengend Snippet: Ceramide profile of GPI anchor-deficient cells. A: Total ceramide (Cer) levels in F21 wild-type and GPI anchor mutants DPM3, PIG-X, PIG-F, and PGAP2. B: Total Cer levels in C311 wild-type and GPI anchor mutant cell lines PIG-O and PIG-U. C: Individual

    Article Snippet: DLPC 12:0/12:0 (850335), PE 17:0/14:1 (PE31:1, LM-1104), PI 17:0/14:1 (PI31:1, LM-1504), PS 17:0/14:1 (PS31:1, LM-1304), C17:0 ceramide (860517), C12:0 SM (860583) and Glucosyl C8:0 Cer (860540) were used as internal lipid standards and were purchased from Avanti Polar Lipids Inc. (Alabaster, AL).

    Techniques: Mutagenesis