Journal: Scientific reports

Article Title: Parallel Reaction Monitoring reveals structure-specific ceramide alterations in the zebrafish.

doi: 10.1038/s41598-019-56466-z

Figure Lengend Snippet: Figure 1. A Parallel Reaction Monitoring-based approach for ceramide quantification. (a) MS2 of d18:1/16:0 ceramide standard in positive ionisation mode. Fragment structures are based on published assignments43,44. (b) Schematic of Parallel Reaction Monitoring. (c) Example PRM chromatograms for C41:1 ceramide from 48 hpf zebrafish embryos, demonstrating presence of multiple LCB-acyl chain isomers with the same m/z. RT: retention time. (d) MS2 spectrum of C41:1 ceramide from 48 hpf zebrafish embryos, the four major LCB fragments are labelled. (d,e) m/z 200-m/z 350, the four major LCB fragments and additional LCB-derived minor fragments are labelled. Unlabelled minor fragments: m/z 224.2371 (d16:1 LCB), m/z 238.2525 (d17:1 LCB).

Article Snippet: Materials. d18:1/16:0 (860516), d18:1/17:0 (860517), d18:1-d7/15:0 (860681), d18:1-d7/18:0 (860677) and d18:1-d7/24:1 (860679) ceramides were purchased from Avanti Polar Lipids.

Techniques: Targeted Proteomics, Derivative Assay

Journal: Journal of Lipid Research

Article Title: Sphingosine 1-phosphate: a novel stimulator of aldosterone secretion

doi: 10.1194/jlr.m500510-jlr200

Figure Lengend Snippet: Fig. 2. S1P stimulates phospholipase D (PLD) activity in ZG cells. ZG cells were labeled with 1 mCi/ml [3H]myristate in DMEM containing 0.2% BSA for 3 h. The cells were then washed three times with this same medium, but in the absence of label, and incubated further in BSA-free DMEM for 2.5 h. Cells were stim- ulated with 5 mM S1P for various times (upper panel) or with increasing concentrations of S1P for 30 min, as indicated (lower panel), in the presence of 1% ethanol. [3H]phosphatidylethanol formation was determined as indicated in Materials and Methods. Results were calculated as a percentage of the radioactivity pres- ent in [3H]phosphatidylethanol compared with that in total lipids and are expressed as fold stimulation relative to incubations with vehicle. Values are means 6 SEM of four independent experi- ments performed in triplicate, except for the value at 5 mM S1P, which is the mean 6 SEM of 15 experiments.

Article Snippet: Sphingosine, S1P, N-acetylsphingosine (C2-ceramide), N-hexanoylsphingosine (C6-ceramide), and phosphatidylethanol were from Avanti Polar Lipids (Alabaster, AL).

Techniques: Activity Assay, Labeling, Incubation, Radioactivity

Journal: Journal of Lipid Research

Article Title: Sphingosine 1-phosphate: a novel stimulator of aldosterone secretion

doi: 10.1194/jlr.m500510-jlr200

Figure Lengend Snippet: Fig. 3. Effect of S1P and angiotensin II on aldosterone secretion and PLD activation in ZG cells. Cells were incubated for 2 h with S1P (5 mM) in the presence (closed bars) or absence (open bars) of angiotensin II (10 nM). Upper panel: Aldosterone secretion was determined as described in Materials and Methods. Results are calculated as described for Fig. 1 and are expressed as means 6 SEM of three independent experiments performed in triplicate. Lower panel: For determination of PLD activity, ethanol (1%) was added 5 min before cell stimulation, and incubations were con- tinued further for 30 min. The enzyme activity was determined by measuring the accumulation of [3H]phosphatidylethanol as described in Materials and Methods. Results were calculated as a percentage of the radioactivity present in [3H]phosphatidylethanol compared with that in total lipids and are expressed as fold stim- ulation relative to control (CTRL) incubations. Values are means 6 SEM of five independent experiments performed in triplicate.

Article Snippet: Sphingosine, S1P, N-acetylsphingosine (C2-ceramide), N-hexanoylsphingosine (C6-ceramide), and phosphatidylethanol were from Avanti Polar Lipids (Alabaster, AL).

Techniques: Activation Assay, Incubation, Activity Assay, Cell Stimulation, Radioactivity, Control

Journal: Journal of Lipid Research

Article Title: Sphingosine 1-phosphate: a novel stimulator of aldosterone secretion

doi: 10.1194/jlr.m500510-jlr200

Figure Lengend Snippet: Fig. 5. Effect of pertussis toxin (PTX) on aldosterone secretion and PLD activation by S1P in ZG cells. Upper panel: Cells were preincubated in the absence (open bars) or presence (closed bars) of 1 mg/ml PTX for 16 h. S1P (5 mM) or vehicle [control (CTRL)] was then added, as indicated, and the incubations were continued further for 2 h. Aldosterone secretion was determined as described in Materials and Methods. Results are calculated and expressed as described for Fig. 1 and are means 6 SEM of three independent experiments performed in triplicate. Lower panel: Cells were preincubated in the absence (open bars) or presence (closed bars) of 1 mg/ml PTX for 16 h. S1P (5 mM) or vehicle [control (CTRL)] was then added, as indicated. Ethanol (1%) was added 5 min before stimulation with S1P, and incubations continued further for 30 min. PLD activity was determined by measuring the accumula- tion of [3H]phosphatidylethanol as described in Materials and Methods. Results were calculated as a percentage of the radioac- tivity present in [3H]phosphatidylethanol compared with that in total lipids and are expressed as fold stimulation relative to control incubations. Values are means 6 SEM of three independent experiments performed in triplicate. ** P , 0.01; * P , 0.05.

Article Snippet: Sphingosine, S1P, N-acetylsphingosine (C2-ceramide), N-hexanoylsphingosine (C6-ceramide), and phosphatidylethanol were from Avanti Polar Lipids (Alabaster, AL).

Techniques: Activation Assay, Control, Activity Assay

Journal: Journal of Lipid Research

Article Title: Sphingosine 1-phosphate: a novel stimulator of aldosterone secretion

doi: 10.1194/jlr.m500510-jlr200

Figure Lengend Snippet: Fig. 8. Involvement of extracellular Ca21 in S1P-induced aldoste- rone secretion and PLD activation. Upper panel: Cells were treated as described for Fig. 1 and preincubated for 30 min with 5 mM EGTA (closed bars), 5 mM BAPTA-AM (gray bars), 5 mM EGTA 1 5 mM BAPTA-AM (hatched bars), or vehicle (open bars). They were then stimulated with vehicle (CTRL) or S1P (5 mM) as indicated and incubated further for 2 h. Aldosterone secretion was determined as described in Materials and Methods. Results are expressed as described for Fig. 1 and are means 6 SEM of three independent experiments. Lower panel: Cells were treated and labeled as described for Fig. 2 and preincubated for 30 min with 5 mM EGTA (closed bars), 5 mM BAPTA-AM (gray bars), 5 mM EGTA 1 5 mM BAPTA-AM (hatched bars), or vehicle (open bars), as indicated. Ethanol (1%) was added 5 min before stimulation with vehicle or S1P (5 mM), and incubations were continued fur- ther for 30 min. PLD activity was determined by measuring the ac- cumulation of [3H]phosphatidylethanol as described in Materials and Methods. Results were calculated and expressed as described for Fig. 2 and are means 6 SEM of three independent experiments. * P , 0.05.

Article Snippet: Sphingosine, S1P, N-acetylsphingosine (C2-ceramide), N-hexanoylsphingosine (C6-ceramide), and phosphatidylethanol were from Avanti Polar Lipids (Alabaster, AL).

Techniques: Activation Assay, Incubation, Labeling, Activity Assay

Journal: Journal of Lipid Research

Article Title: Sphingosine 1-phosphate: a novel stimulator of aldosterone secretion

doi: 10.1194/jlr.m500510-jlr200

Figure Lengend Snippet: Fig. 9. Involvement of PKC in PLD activation by S1P in ZG cells. Upper panel: Cells were preincubated with vehicle (open bars) or 2 mM PMA for 48 h (closed bars) to downregulate PKC. Ethanol (1%) was added 5 min before stimulation with S1P (5 mM), PMA (2 mM), or vehicle, and incubations were continued further for 30 min. PLD activity was determined by measuring the accumula- tion of [3H]phosphatidylethanol as described in Materials and Methods. Results were calculated as a percentage of the radioac- tivity present in [3H]phosphatidylethanol compared with that in total lipids and are expressed as fold stimulation relative to con- trol (CTRL) incubations. Values are means 6 SEM of three inde- pendent experiments performed in triplicate. Lower panel: Cells were preincubated for 30 min with vehicle (CTRL), 2 mM hispidine (HISP), 0.5 mM dequalinium (DEQ), 5 mM rottlerin (ROT), or 0.5 mM dequalinium 1 5 mM rottlerin (DEQ1ROT), as indicated. Ethanol (1%) was added 5 min before stimulation with 5 mM S1P or vehicle, and incubations were continued further for 30 min. Results were calculated and expressed as for the upper panel and are means 6 SEM of three independent experiments performed in triplicate. ** P , 0.01; * P , 0.05;1 P , 0.05, DEQ1ROT versus DEQ or ROT.

Article Snippet: Sphingosine, S1P, N-acetylsphingosine (C2-ceramide), N-hexanoylsphingosine (C6-ceramide), and phosphatidylethanol were from Avanti Polar Lipids (Alabaster, AL).

Techniques: Activation Assay, Activity Assay

Journal: Journal of Lipid Research

Article Title: Sphingosine 1-phosphate: a novel stimulator of aldosterone secretion

doi: 10.1194/jlr.m500510-jlr200

Figure Lengend Snippet: Fig. 11. Inhibition of S1P-stimulated PLD and aldosterone secre- tion by N-hexanoylsphingosine (C6-ceramide) in ZG cells. Upper panel: Cells were treated and labeled as described for Fig. 3. They were then preincubated in the absence (open bars) or presence (closed bars) of 10 mM C6-ceramide for 2 h in BSA-free DMEM. S1P (5 mM) was added, and incubations were continued further for 30 min in the presence of 1% ethanol. PLD activity was determined by measuring the accumulation of [3H]phosphatidylethanol as described in Materials and Methods. Results were calculated as a percentage of the radioactivity present in [3H]phosphatidylethanol compared with that in total lipids and expressed as fold stimula- tion relative to control (CTRL) incubations. Values are means 6 SEM of four independent experiments performed in triplicate. Lower panel: Cells were preincubated with vehicle (open circles) or with the indicated concentrations of C6-ceramide (closed circles) for 2 h in DMEM supplemented with 0.2% BSA. S1P (5 mM) was then added, and incubations were continued further for 2 h. Aldosterone secretion was determined as described in Materials and Methods. Results are expressed as fold stimulation relative to incubations with vehicle and are means 6 SEM of four indepen- dent experiments performed in triplicate. ** P , 0.01.

Article Snippet: Sphingosine, S1P, N-acetylsphingosine (C2-ceramide), N-hexanoylsphingosine (C6-ceramide), and phosphatidylethanol were from Avanti Polar Lipids (Alabaster, AL).

Techniques: Inhibition, Labeling, Activity Assay, Radioactivity, Control

Journal: The FASEB Journal

Article Title: ORMDL Proteins Turnover via Proteasome and Autophagy Is Cell‐Type Dependent and Tied to Ceramide Homeostasis

doi: 10.1096/fj.202502924RR

Figure Lengend Snippet: Regulation of ORMDLs stability in human RPE‐1 cells and mouse BMMCs. (A) Levels of ORMDLs, SPTLC1, SPTLC2, and total d18:1 ceramides (measured by LC‐ESI‐MS/MS) were assessed in human RPE‐1 cells and mouse BMMCs after 24‐h treatment with 10 μM myriocin or 10 μM FB 1 , and compared with untreated controls. (B) Quantification of ORMDLs, SPTLC1, SPTLC2, and the LC3‐II/LC3‐I ratio was performed in untreated RPE‐1 cells and BMMCs, and compared with cells treated for 24 h with 50 μM CQ or 10 μM MG132, as determined by immunoblotting. (C) Effects of p97/VCP inhibition by CB‐5083 on ORMDLs, SPTLC1, SPTLC2, the LC3‐II/LC3‐I ratio, ATF4, and p62 levels were assessed in RPE‐1 cells and BMMCs. Protein levels in (A‐C) were normalized to total protein loading using Ponceau S staining. Data are presented as geometric mean ± GSEM ( N = 4). Statistical analysis was performed using one‐way ANOVA with Tukey's post hoc test. Significance levels: p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), p < 0.0001 (****); n.s., not significant.

Article Snippet: Sphingolipid concentrations of S1P, dhC1P, and C1P were determined by single‐point calibration using external standards [ ]: 50 ng S1P d18:1 (Cat# 860492), 50 ng dhC1P d18:0/C16:0 (Cat# 860522), and 50 ng C1P d18:1/C16:0 (Cat# 860533), all from Avanti Polar Lipids.

Techniques: Tandem Mass Spectroscopy, Western Blot, Inhibition, Staining