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: The FASEB Journal

Article Title: The NMR‐based characterization of the FTY720‐SET complex reveals an alternative mechanism for the attenuation of the inhibitory SET‐PP2A interaction

doi: 10.1096/fj.201802264r

Figure Lengend Snippet: Figure 3. FTY720 binds SET similarly as D-e-C18 ceramide. A) Overlaid [1H-1H] total correlational spectroscopy spectra of FTY720. Quantitation of line broadening as a function of peak volume and increasing concentration of Nd-SETWT (0–50 mM). Chemical structure of functional groups on FTY720 affected by the addition of Nd-SET. B) CSP of NdCd-SET induced by 3 mM (black bars), 7 mM (red bars), and 10 mM FTY720 (blue bars) as a function of assigned residue. CSP titrations were performed in triplicate. Red line is calculated as 3s, and any value .3s is considered a significant shift. C) Model of FTY720 bound SET. Residues colored red underwent a significant chemical shift during NMR titration analysis. D) Association between endogenous SET, FLAG-SETWT, FLAG-SETE111A, and FLAG-SETR71A mutants and PP2AC 6 5 mM FTY720 was monitored by PLA using antibodies against SET, FLAG, or PP2AC. SET mutants were generated from CSP and modeling. Representative images of n $ 3 independent experiments per group. Scale bar, 100 mm. E) Quantitation of PLA for SET mutants designed to limit response to FTY720 treatment. Data are means 6 SEM of n $3 independent experiments per group, analyzed by 2-way ANOVA with Tukey’s post hoc test. *P , 0.05, ***P , 0.001.

Article Snippet: Brinkmann, V., Billich, A., Baumruker, T., Heining, P., Schmouder, R., Francis, G., Aradhye, S., and Burtin, P. (2010) Fingolimod (FTY720): discovery and development of an oral drug to treat multiple sclerosis.

Techniques: Spectroscopy, Quantitation Assay, Concentration Assay, Functional Assay, Residue, Titration, Generated

Journal: The FASEB Journal

Article Title: The NMR‐based characterization of the FTY720‐SET complex reveals an alternative mechanism for the attenuation of the inhibitory SET‐PP2A interaction

doi: 10.1096/fj.201802264r

Figure Lengend Snippet: Figure 4. Lipid binding may alter SET dimerization domain to prevent oligomerization. A) Spectrum of NdCd highlights peaks that undergo line broadening upon the addition of FTY720. These peaks correspond to residues in the dimerization domain of SET. Blowout highlights broadened peaks (red) at 10 mM FTY720. B) Quantitation of the total change in peak height of assigned (continued on next page)

Article Snippet: Brinkmann, V., Billich, A., Baumruker, T., Heining, P., Schmouder, R., Francis, G., Aradhye, S., and Burtin, P. (2010) Fingolimod (FTY720): discovery and development of an oral drug to treat multiple sclerosis.

Techniques: Binding Assay, Quantitation Assay

Journal: The FASEB Journal

Article Title: The NMR‐based characterization of the FTY720‐SET complex reveals an alternative mechanism for the attenuation of the inhibitory SET‐PP2A interaction

doi: 10.1096/fj.201802264r

Figure Lengend Snippet: Figure 6. Reconstitution of PP2ACa and B56g expression restores cellular response to FTY720. A) Interaction between endogenous SET and B56g 6 5 mM FTY720, 5 mM D-e-C18 ceramide, or 1 mM OP449 treatment for 3 h assessed by PLA using antibodies against SET and B56g. Quantitation of PLA experiments. Error bars are SEM of n $3 independent experiments per group. Data are means 6

Article Snippet: Brinkmann, V., Billich, A., Baumruker, T., Heining, P., Schmouder, R., Francis, G., Aradhye, S., and Burtin, P. (2010) Fingolimod (FTY720): discovery and development of an oral drug to treat multiple sclerosis.

Techniques: Expressing, Quantitation Assay

Journal: The FASEB Journal

Article Title: The NMR‐based characterization of the FTY720‐SET complex reveals an alternative mechanism for the attenuation of the inhibitory SET‐PP2A interaction

doi: 10.1096/fj.201802264r

Figure Lengend Snippet: Figure 7. Myosin IIa is a potential target of FTY720-activated PP2A. A) Validation of PP2AC/myosin IIa association observed in SILAC data by coimmunoprecipitation. Exogenous expression of HA-PP2ACaWT in A549 cells using magnetic HA-conjugated beads 6 treatment of 20 mM FTY720 for 2 h. B) Association between PP2AC and myosin IIa in shSCR and shB56g stable knockdown cell lines 6 treatment of 5 mM FTY720 for 3 h by PLA using antibodies against PP2AC and myosin IIa. Quantitation of PLA experiments. Data are means 6 SEM of n = 3 independent experiments, analyzed by 2-way ANOVA with Tukey’s post hoc test. *P , 0.05, **P , 0.01. C) PP2A activity assay in shSCR and shB56g stable knockdown cell lines 6 treatment of 5 mM FTY720 for 16 h using purified rabbit MYH9 as a substrate. Data are means 6 SD of n = 3 independent experiments analyzed by 2-way ANOVA (continued on next page)

Article Snippet: Brinkmann, V., Billich, A., Baumruker, T., Heining, P., Schmouder, R., Francis, G., Aradhye, S., and Burtin, P. (2010) Fingolimod (FTY720): discovery and development of an oral drug to treat multiple sclerosis.

Techniques: Biomarker Discovery, Multiplex sample analysis, Expressing, Knockdown, Quantitation Assay, Activity Assay

Journal: The FASEB Journal

Article Title: The NMR‐based characterization of the FTY720‐SET complex reveals an alternative mechanism for the attenuation of the inhibitory SET‐PP2A interaction

doi: 10.1096/fj.201802264r

Figure Lengend Snippet: Figure 8. Phosphorylation of SET influences PP2A activity. A) Association between endogenous SET, FLAG-SETWT, FLAG- SETS171A, and FLAG-SETS171E, mutants, and PP2AC 6 5 mM FTY720 with a pCDH empty vector control measured by PLA using antibodies against SET, FLAG, and PP2AC. Data are means 6 SEM of n = 3 independent experiments, analyzed by 2-way ANOVA with Tukey’s post hoc test. *P , 0.05, **P , 0.01, ****P , 0.0001. B) Association between FLAG-SETS171A and FLAG-SETS171E and B56g 6 5 mM FTY720 assessed by PLA using antibodies against FLAG and B56g. Data are means 6 SEM of n = 3 independent experiments analyzed by 2-way ANOVA with Tukey’s post hoc test. *P , 0.05, **P , 0.01. C) Western blot of PP2A-p-Tyr307 of PP2AC as a marker of activity normalized to b-actin and FLAG expression. Representative blot of n = 3 individual experiments. D) Blots were quantified with ImageJ. Data are means normalized to actin and FLAG 6 SD and analyzed by 2-way ANOVA with Tukey’s post hoc test. *P , 0.05. E) Quantification of GA cross-linking of FLAG-SETWT, FLAG-SETS171A, and FLAG-SETS171E

Article Snippet: Brinkmann, V., Billich, A., Baumruker, T., Heining, P., Schmouder, R., Francis, G., Aradhye, S., and Burtin, P. (2010) Fingolimod (FTY720): discovery and development of an oral drug to treat multiple sclerosis.

Techniques: Phospho-proteomics, Activity Assay, Plasmid Preparation, Control, Western Blot, Marker, 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 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: