Journal: Traffic (Copenhagen, Denmark)

Article Title: ENDOCYTOSIS OF LIGAND ACTIVATED SPHINGOSINE 1-PHOSPHATE RECEPTOR 1 MEDIATED BY THE CLATHRIN-PATHWAY

doi: 10.1111/tra.12343

Figure Lengend Snippet: (A-D) HeLa cells not expressing (A) or transiently expressing 3xHA-S1PR1-eGFP (B-D) were incubated with anti-HA antibody to label receptor at the cell surface and then follow their internalization upon incubation with DMSO only (Carrier) or the S1PR1 ligands (S1P or FTY720P). Fluorescent transferrin-Alexa Fluor 647 was used to monitor the clathrin-based receptor mediated endocytosis of transferrin. HA antibody was used to follow the uptake of S1P1R. Images for each condition correspond to a center plane obtained using spinning disc confocal microscopy from which quantification data was obtained. An acid wash step at the end of the experiment was used to remove the surface bound transferrin or HA antibodies. Scale bar 10 μm.

Article Snippet: Reagents Sphingosine-1-phosphate (S1P) and FTY720-phosphate (FTY720P) were from Echelon Bioscience (Salt Lake City, UT), poly-d-lysine (Sigma) and collagen (BD Biosciences).

Techniques: Expressing, Incubation, Confocal Microscopy

Journal: Traffic (Copenhagen, Denmark)

Article Title: ENDOCYTOSIS OF LIGAND ACTIVATED SPHINGOSINE 1-PHOSPHATE RECEPTOR 1 MEDIATED BY THE CLATHRIN-PATHWAY

doi: 10.1111/tra.12343

Figure Lengend Snippet: (A) Effect of clathrin or AP2 depletion in the uptake of S1PR1 in Hela cells transiently expressing 3xHA-S1P1-eGFP. Cells were depleted of clathrin or of its endocytic adaptor AP2 by transduction with lentiviruses encoding shRNA specific for clathrin heavy chain (CLC) or the μ2 subunit of AP2 (AP2); scrambled shRNA was used as a negative control. The cells were incubated with anti-HA antibody to follow the uptake of S1PR1 upon addition of DMSO only (Carrier) or the ligands (S1P or FTY720P). Transferrin-Alexa Fluor 647 was used to monitor its clathrin-based receptor mediated endocytosis. The representative images correspond to a center plane from which quantification data was obtained. An acid wash step at the end of the experiment was used to remove surface bound transferrin and anti-HA antibodies. Scale bar 10 μm.

Article Snippet: Reagents Sphingosine-1-phosphate (S1P) and FTY720-phosphate (FTY720P) were from Echelon Bioscience (Salt Lake City, UT), poly-d-lysine (Sigma) and collagen (BD Biosciences).

Techniques: Expressing, Transduction, shRNA, Negative Control, Incubation

Journal: Traffic (Copenhagen, Denmark)

Article Title: ENDOCYTOSIS OF LIGAND ACTIVATED SPHINGOSINE 1-PHOSPHATE RECEPTOR 1 MEDIATED BY THE CLATHRIN-PATHWAY

doi: 10.1111/tra.12343

Figure Lengend Snippet: (A) MEF cells from mice expressing (β-Arrestin1/2+/+) or not (β-Arrestin1/2−/−) β-arrestin1 and β-arrestin2 and stably expressing 3xHA-S1PR1-eGFP were incubated with anti-HA antibody in the presence of DMSO only (Carrier) or the ligands S1P or FTY720P. Images for each condition correspond to a center plane obtained using spinning disc confocal microscopy. An acid wash step at the end of the experiment was used to remove most of the surface bound anti-HA antibodies. Scale bar 10 μm.

Article Snippet: Reagents Sphingosine-1-phosphate (S1P) and FTY720-phosphate (FTY720P) were from Echelon Bioscience (Salt Lake City, UT), poly-d-lysine (Sigma) and collagen (BD Biosciences).

Techniques: Expressing, Stable Transfection, Incubation, Confocal Microscopy

Journal: Traffic (Copenhagen, Denmark)

Article Title: ENDOCYTOSIS OF LIGAND ACTIVATED SPHINGOSINE 1-PHOSPHATE RECEPTOR 1 MEDIATED BY THE CLATHRIN-PATHWAY

doi: 10.1111/tra.12343

Figure Lengend Snippet: HEK 293A cells stably expressing 3xHA-S1PR1-mCherry were incubated or not with anti-HA antibody to label receptor at the cell surface and then follow their internalization upon incubation with DMSO only (carrier) or the S1PR1 ligands (S1P or FTY720P). Fluorescent transferrin-Alexa Fluor 647 was used to monitor the clathrin-based receptor mediated endocytosis of transferrin. Data obtained using flow cytometry was from more than 10,000 cells analyzed per condition.

Article Snippet: Reagents Sphingosine-1-phosphate (S1P) and FTY720-phosphate (FTY720P) were from Echelon Bioscience (Salt Lake City, UT), poly-d-lysine (Sigma) and collagen (BD Biosciences).

Techniques: Stable Transfection, Expressing, Incubation, Flow Cytometry

Journal: Traffic (Copenhagen, Denmark)

Article Title: ENDOCYTOSIS OF LIGAND ACTIVATED SPHINGOSINE 1-PHOSPHATE RECEPTOR 1 MEDIATED BY THE CLATHRIN-PATHWAY

doi: 10.1111/tra.12343

Figure Lengend Snippet: (A) HeLa cells transiently expressing 3xHA-S1PR1-eGFP were incubated with anti-HA antibody in the absence (− dynasore-OH) or presence (+ dynasore-OH) of the modified more potent cell permeable inhibitor of dynamin, dynasore-OH. Following brief 15 min incubation with dynasore-OH, the media was replaced for 30 min with a new solution only containing DMSO, S1P or FTY720P. Fluorescent transferrin-Alexa Fluor 647 was used to follow the efficiency of the clathrin-based endocytic pathway. Images obtained using spinning disc confocal microscopy correspond to a center plane from which quantification data was obtained. An acid wash step at the end of the experiment was used to remove the surface bound transferrin and anti-HA antibodies. Scale bar, 10 μm.

Article Snippet: Reagents Sphingosine-1-phosphate (S1P) and FTY720-phosphate (FTY720P) were from Echelon Bioscience (Salt Lake City, UT), poly-d-lysine (Sigma) and collagen (BD Biosciences).

Techniques: Expressing, Incubation, Modification, Confocal Microscopy

Journal: Scientific reports

Article Title: Effect of FTY720P on lipid accumulation in HEPG2 cells.

doi: 10.1038/s41598-023-46011-4

Figure Lengend Snippet: Figure 2. S1P and FTY720P induce lipid accumulation in HepG2 cells. Lipid accumulation in the presence of (a) Sphk1 inhibitor PF543; (b) blockers of all S1PRs. Values are means ± SEM. N = 4. Bars not sharing a common letter are considered significantly different from each other at P < 0.05; c) various concentrations of the S1P analog FTY720P. Values are means ± SEM; N = 3. *Significantly different from the control at P < 0.05.

Article Snippet: FTY720P (Cat # sc-205332A), rosiglitazone (Cat # sc-202795), anti-SREBP1 (Cat# sc-365513, Lot#B1821), anti-p-mTOR (Cat# sc-293132, Lot#K2717), anti-mTOR (Cat# sc-8319, Lot#K2415), anti-GAPDH (Cat# sc-47724, Lot# H0917) mouse primary antibodies as well as anti-mouse horse radish peroxidase (HRP) conjugated secondary antibodies (Cat# sc-2005, Lot# C2011) were purchased all from Santa Cruz Biotechnology, CA, USA.

Techniques: Control

Journal: Scientific reports

Article Title: Effect of FTY720P on lipid accumulation in HEPG2 cells.

doi: 10.1038/s41598-023-46011-4

Figure Lengend Snippet: Figure 3. Effect of FTY720P on lipid accumulation in the presence of (a) S1PR1 blocker W146. (b) S1PR2 blocker JTE 013. (c) S1PR4 blocker CYM50358. Values are means ± SEM; N = 5. Bars not sharing a common letter are considered significantly different from each other at P < 0.05.

Article Snippet: FTY720P (Cat # sc-205332A), rosiglitazone (Cat # sc-202795), anti-SREBP1 (Cat# sc-365513, Lot#B1821), anti-p-mTOR (Cat# sc-293132, Lot#K2717), anti-mTOR (Cat# sc-8319, Lot#K2415), anti-GAPDH (Cat# sc-47724, Lot# H0917) mouse primary antibodies as well as anti-mouse horse radish peroxidase (HRP) conjugated secondary antibodies (Cat# sc-2005, Lot# C2011) were purchased all from Santa Cruz Biotechnology, CA, USA.

Techniques:

Journal: Scientific reports

Article Title: Effect of FTY720P on lipid accumulation in HEPG2 cells.

doi: 10.1038/s41598-023-46011-4

Figure Lengend Snippet: Figure 4. S1PR3 and Gq mediate the FTY720P effect on lipid accumulation. (a) Effect of FTY720P on lipid accumulation in the presence of S1PR3 blocker CAY10444. (b) Effect of FTY720P on lipid accumulation in the presence of the Gq inhibitor YM254890. (c) Protein expression of S1PR3. The blot is representative of an experiment repeated 3 times. Values were normalized to GAPDH and reported as arbitrary densitometry units. All values are means ± SEM; N = 3. Bars not sharing a common letter are considered significantly different from each other at P < 0.05.

Article Snippet: FTY720P (Cat # sc-205332A), rosiglitazone (Cat # sc-202795), anti-SREBP1 (Cat# sc-365513, Lot#B1821), anti-p-mTOR (Cat# sc-293132, Lot#K2717), anti-mTOR (Cat# sc-8319, Lot#K2415), anti-GAPDH (Cat# sc-47724, Lot# H0917) mouse primary antibodies as well as anti-mouse horse radish peroxidase (HRP) conjugated secondary antibodies (Cat# sc-2005, Lot# C2011) were purchased all from Santa Cruz Biotechnology, CA, USA.

Techniques: Expressing

Journal: Scientific reports

Article Title: Effect of FTY720P on lipid accumulation in HEPG2 cells.

doi: 10.1038/s41598-023-46011-4

Figure Lengend Snippet: Figure 5. Effect of FTY720P on lipid accumulation in the presence of (a) the PI3K inhibitor wortmannin. (b) the mTOR inhibitor rapamycin. All values are means ± SEM; N = 4. Bars not sharing a common letter are considered significantly different from each other at P < 0.05.

Article Snippet: FTY720P (Cat # sc-205332A), rosiglitazone (Cat # sc-202795), anti-SREBP1 (Cat# sc-365513, Lot#B1821), anti-p-mTOR (Cat# sc-293132, Lot#K2717), anti-mTOR (Cat# sc-8319, Lot#K2415), anti-GAPDH (Cat# sc-47724, Lot# H0917) mouse primary antibodies as well as anti-mouse horse radish peroxidase (HRP) conjugated secondary antibodies (Cat# sc-2005, Lot# C2011) were purchased all from Santa Cruz Biotechnology, CA, USA.

Techniques:

Journal: Scientific reports

Article Title: Effect of FTY720P on lipid accumulation in HEPG2 cells.

doi: 10.1038/s41598-023-46011-4

Figure Lengend Snippet: Figure 6. Effect of FTY720P on lipid accumulation in the presence of (a) the SREBP inhibitor fatostatin. (b) The PPARγ inhibitor GW9662. Values are means ± SEM; N = 5. Bars not sharing a common letter are considered significantly different from each other at P < 0.05.

Article Snippet: FTY720P (Cat # sc-205332A), rosiglitazone (Cat # sc-202795), anti-SREBP1 (Cat# sc-365513, Lot#B1821), anti-p-mTOR (Cat# sc-293132, Lot#K2717), anti-mTOR (Cat# sc-8319, Lot#K2415), anti-GAPDH (Cat# sc-47724, Lot# H0917) mouse primary antibodies as well as anti-mouse horse radish peroxidase (HRP) conjugated secondary antibodies (Cat# sc-2005, Lot# C2011) were purchased all from Santa Cruz Biotechnology, CA, USA.

Techniques:

Journal: Scientific reports

Article Title: Effect of FTY720P on lipid accumulation in HEPG2 cells.

doi: 10.1038/s41598-023-46011-4

Figure Lengend Snippet: Figure 13. The signaling pathway activated by FTY720P.

Article Snippet: FTY720P (Cat # sc-205332A), rosiglitazone (Cat # sc-202795), anti-SREBP1 (Cat# sc-365513, Lot#B1821), anti-p-mTOR (Cat# sc-293132, Lot#K2717), anti-mTOR (Cat# sc-8319, Lot#K2415), anti-GAPDH (Cat# sc-47724, Lot# H0917) mouse primary antibodies as well as anti-mouse horse radish peroxidase (HRP) conjugated secondary antibodies (Cat# sc-2005, Lot# C2011) were purchased all from Santa Cruz Biotechnology, CA, USA.

Techniques:

Journal: PLoS ONE

Article Title: Sphingosine 1-Phosphate Modulates Antigen Capture by Murine Langerhans Cells via the S1P 2 Receptor Subtype

doi: 10.1371/journal.pone.0049427

Figure Lengend Snippet: XS52 cells were incubated with FITC-labeled dextran in the presence or absence of S1P (5 µM), FTY720-P (1 µM), VPC24191 (10 µM), and SEW2871 (1 µM) for 15 min. Fluorescence intensity of cells was analyzed by flow cytometry and relative endocytosis was calculated. Data are expressed as the mean ± SEM of results from at least three independent experiments. **P < 0.01 indicate a statistically significant difference vs. control experiments (A). Cells were treated with similar concentrations of S1P, FTY720-P, VPC24191, and SEW2871 for 15 min followed by the detection of Akt activity (B). Values of the densitometric analysis are expressed as x-fold decrease of p-Akt formation compared to untreated cells ± SEM from three experiments. **P<0.01 indicates a statistically significant difference versus control (B).

Article Snippet: SEW2871 and FTY720-phosphate (FTY720-P) were obtained from Cayman Chemical (Michigan, USA).

Techniques: Incubation, Labeling, Fluorescence, Flow Cytometry, Control, Activity Assay