ceramide  (Avanti Polar)

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Cell density-dependent membrane distribution of ganglioside GM3 in melanoma cells

doi: 10.1007/s00018-023-04813-9

Figure Lengend Snippet: Binding of anti-GM3 antibody (GMR6) to GM3/partner lipid is dependent on incubation temperatures and phase transition temperatures of partner lipids. A Binding of 0.37 µg/mL of anti-GM3 antibody to indicated lipids (0.5 nmol/well) was measured by ELISA. GM1, ganglioside GM1; GM2, ganglioside GM2; GM3, ganglioside GM3; GlcCer, glucosylceramide; LacCer, lactosylceramide; DMPC, 1,2-dimyristoyl-sn-glycero-3-phosphocholine; DPPC, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine; DOPC, 1,2-dioleoyl-sn-glycero-3-phosphocholine; POPC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; pSM, palmitoyl sphingomyelin;Cer, ceramide; DPPE, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine; DOPE, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine; DOPS, 1,2-dioleoyl-sn-glycero-3-phospho-L-serine; DOPG, 1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol); TOCL, 1',3'-bis[1,2-dioleoyl-sn-glycero-3-phospho]-glycerol; CerPE, N-acyl-sphingosylphosphorylethanolamine. B Binding of 0.37 µg/mL of anti-GM3 antibody to GM3/DOPC (1:9), GM3/POPC (1:9), GM3/DMPC (1:9) or GM3/DPPC (1:9) (0.5 nmol GM3/well) at indicated temperature measured by ELISA. (C) Binding of 0.37 µg/mL of anti-GM3 antibody to GM3/pSM (1:9), GM3/GlcCer (1:9), GM3/GM1 (1:9) or GM3 alone (0.5 nmol GM3/well) at indicated temperatures measured by ELISA. Data in A–C are means ± SD of three experiments. D Binding of anti-GM3 antibody (10 ng/mL) to GM3/DOPC (1:5) (orange) or GM3/DPPC (1:5) (green) liposomes measured by surface plasmon resonance (SPR). SPR was performed as described in Methods. E Binding of anti-GM3 antibody to DPPC/GM3 (5:2) (orange) or DPPC/GM3/Chol (5:2:5) (green) liposomes. F Binding of different concentration of anti-GM3 antibody to GM3/DPPC (1:9) liposomes

Article Snippet: GM2 (GalNAcβ1,4(Neu5Acα2,3)Galβ1,4Glcβ1,1’-ceramide) from bovine brain was from Wako Pure Chemical Industries (Osaka, Japan).

Techniques: Binding Assay, Incubation, Sublimation, Enzyme-linked Immunosorbent Assay, Liposomes, SPR Assay, Concentration Assay

Journal: Cell Communication and Signaling : CCS

Article Title: Ceramide from sphingomyelin hydrolysis induces neuronal differentiation, whereas de novo ceramide synthesis and sphingomyelin hydrolysis initiate apoptosis after NGF withdrawal in PC12 Cells

doi: 10.1186/s12964-021-00767-2

Figure Lengend Snippet: The effect of NGF on differentiation in PC12 and TrkA cells. PC12 ( A – E ) and TrkA ( F – J ) cells were induced to differentiate with 0.05 ng/ml NGF. Neuronal morphology (shrunken soma and long extended neurites that synapse on other cells) is indicated with arrows (×400 magnification). PC12 ( K ) and TrkA ( L ) cells were treated as above, lipids were extracted and ceramide and SM levels quantified by the DG kinase assay and SM mass assay as described in Materials and Methods. Each value represents the mean of duplicate determinations from at least three experiments; bars, SEM

Article Snippet: 30 μL ceramide was loaded into each lane of a TLC plate (6 nm silica gel plates of 0.25 thickness from Whatman) along with known concentrations of ceramide (natural ceramide type III from bovine brain from Sigma-Aldrich, St. Louis, MO) as described [ ].

Techniques: Kinase Assay, Mass Assay

Journal: Cell Communication and Signaling : CCS

Article Title: Ceramide from sphingomyelin hydrolysis induces neuronal differentiation, whereas de novo ceramide synthesis and sphingomyelin hydrolysis initiate apoptosis after NGF withdrawal in PC12 Cells

doi: 10.1186/s12964-021-00767-2

Figure Lengend Snippet: A Time course for ceramide generation in PC12 cells treated as above. Lipids were extracted and ceramide levels quantified by the DG kinase assay as described in Materials and Methods. B Time course for SM generation in PC12 cells treated as above. Lipids were extracted and SM levels quantified by the SM mass assay as described in Materials and Methods. C Time course for ceramide generation in TrkA cells treated as above. Lipids were extracted and ceramide levels quantified by the DG kinase assay as described in Materials and Methods. D Time course for SM generation in TrkA cells treated as above. Lipids were extracted and SM levels quantified by the SM mass assay as described in Materials and Methods. Each value represents the mean of duplicate determinations from at least three experiments; bars, SEM; ** p ≤ 0.01

Article Snippet: 30 μL ceramide was loaded into each lane of a TLC plate (6 nm silica gel plates of 0.25 thickness from Whatman) along with known concentrations of ceramide (natural ceramide type III from bovine brain from Sigma-Aldrich, St. Louis, MO) as described [ ].

Techniques: Kinase Assay, Mass Assay

Journal: Cell Communication and Signaling : CCS

Article Title: Ceramide from sphingomyelin hydrolysis induces neuronal differentiation, whereas de novo ceramide synthesis and sphingomyelin hydrolysis initiate apoptosis after NGF withdrawal in PC12 Cells

doi: 10.1186/s12964-021-00767-2

Figure Lengend Snippet: The effect of desipramine on differentiation of PC12 and TrkA cells. A Time course for ceramide generation in PC12 cells treated as above. Lipids were extracted and ceramide levels quantified by the DG kinase assay as described in Materials and Methods. B Time course for SM generation in PC12 cells treated as above. Lipids were extracted and SM levels quantified by the SM mass assay as described in Materials and Methods. C Time course for ceramide generation in TrkA cells treated as above. Lipids were extracted and ceramide levels quantified by the DG kinase assay as described in Materials and Methods. D Time course for SM generation in TrkA cells treated as above. Lipids were extracted and SM levels quantified by the SM mass assay as described in Materials and Methods. Each value represents the mean of duplicate determinations from at least three experiments; bars, SEM; ** p ≤ 0.01

Article Snippet: 30 μL ceramide was loaded into each lane of a TLC plate (6 nm silica gel plates of 0.25 thickness from Whatman) along with known concentrations of ceramide (natural ceramide type III from bovine brain from Sigma-Aldrich, St. Louis, MO) as described [ ].

Techniques: Kinase Assay, Mass Assay

Journal: Cell Communication and Signaling : CCS

Article Title: Ceramide from sphingomyelin hydrolysis induces neuronal differentiation, whereas de novo ceramide synthesis and sphingomyelin hydrolysis initiate apoptosis after NGF withdrawal in PC12 Cells

doi: 10.1186/s12964-021-00767-2

Figure Lengend Snippet: Response to NGF withdrawal in PC12 or TrkA cells. Differentiated PC12 (UPPER) and Trk A (LOWER) cells were deprived of NGF for up to 48 h and collected every 3–6 h for viability assay as well as extraction and quantification of lipids. Quantification of apoptosis (viability) was performed by trypan blue staining of dead cells as well as by staining the cells with the DNA specific fluorochrome Hoechst 33,258 as described in Materials and Methods. A minimum of 250 cells was scored for the incidence of apoptosis in each experiment. Endogenous ceramide was quantified by DAG kinase assay and sphingomyelin by SM mass assay as described in Materials and Methods. Each value represents the mean of duplicate determinations from at least three independent experiments. bars, SEM

Article Snippet: 30 μL ceramide was loaded into each lane of a TLC plate (6 nm silica gel plates of 0.25 thickness from Whatman) along with known concentrations of ceramide (natural ceramide type III from bovine brain from Sigma-Aldrich, St. Louis, MO) as described [ ].

Techniques: Viability Assay, Staining, Kinase Assay, Mass Assay

Journal: Cell Communication and Signaling : CCS

Article Title: Ceramide from sphingomyelin hydrolysis induces neuronal differentiation, whereas de novo ceramide synthesis and sphingomyelin hydrolysis initiate apoptosis after NGF withdrawal in PC12 Cells

doi: 10.1186/s12964-021-00767-2

Figure Lengend Snippet: The effect of specific ceramide inhibition on PC12 or TrkA cells following NGF removal. Differentiated PC12 and TrkA cells were deprived of NGF for 24 h in the presence of either 15 μM desipramine or 25 μM FB 1 . Control cells treated with vehicle or inhibitors alone are in white, cells deprived of NGF are depicted with hatching. Each value represents the mean of duplicate determinations from three independent experiments; bars, SEM; * p < 0.05, ** p << 0.05, *** p << 0.01. A Measurement of ceramide levels. Lipids were extracted and quantified by DAG kinase assay as described in Materials and Methods. B Measurement of SM levels. Lipids were extracted and quantified by SM mass assay as described in Materials and Methods. C Measurement of viability. A minimum of 250 cells was scored by trypan blue exclusion for the incidence of apoptosis in each experiment

Article Snippet: 30 μL ceramide was loaded into each lane of a TLC plate (6 nm silica gel plates of 0.25 thickness from Whatman) along with known concentrations of ceramide (natural ceramide type III from bovine brain from Sigma-Aldrich, St. Louis, MO) as described [ ].

Techniques: Inhibition, Kinase Assay, Mass Assay