Journal: Science Advances

Article Title: Structure of S1PR2–heterotrimeric G 13 signaling complex

doi: 10.1126/sciadv.abn0067

Figure Lengend Snippet: ( A ) Dose-response curves of S1PR2 and S1PR3 for the TGFα shedding assay using FTY720-P. The chemical structure of FTY720-P is shown. Data are means ± SD ( n = 3). The membrane expression of S1PRs is shown in figs. S4A and S6C. EV, empty vector. ( B ) JTE-013 reduces the FTY720-P–induced activation of S1PR2 but not S1PR3. The chemical structure of JTE-013 is shown. Data are means ± SD ( n = 3). ( C ) Structural comparisons among S1PRs imply the unique roles of residue F274 of S1PR2. The residues are shown in sticks. ( D ) The sequence alignment of S1PR2-TM7 from different species. The conserved residues are highlighted. The residue F274 is indicated by a star. ( E and G ) Mutagenesis analysis of S1PR2 F274I for S1P (E) and FTY720-P (G). The representative dose-response curves from same-day experiment are shown. Data are means ± SD ( n = 3). ( F and H ) RAi of each varient for S1P (F) and FTY720-P (H). Each LogRAi plot denotes a single measurement from a dose-response experiment. Data are means ± SD ( n = 3 to 5 independent experiments). * P < 0.05, *** P < 0.001, and **** P < 0.0001; mutant versus wild-type values according to one-way ANOVA with Dunnett’s multiple comparison test.

Article Snippet: The ligand S1P (Tocris) or FTY720 (S)-phosphate (FTY720-P, Echelon) was serially diluted in HBSS supplemented with 5 mM Hepes (pH 7.4) and 0.01% fatty acid–free bovine serum albumin (BSA; GoldBio) to prepare 10× stock solutions.

Techniques: Expressing, Plasmid Preparation, Activation Assay, Sequencing, Mutagenesis

Journal: Science Advances

Article Title: Structure of S1PR2–heterotrimeric G 13 signaling complex

doi: 10.1126/sciadv.abn0067

Figure Lengend Snippet: ( A ) The immunofluorescence staining of HEK293 cells that express S1PR2 or its variants after vehicle or 1 μM ligand treatment. Scale bar, 10 μm. DAPI, 4′,6-diamidino-2-phenylindole. ( B ) Internalization of S1PR2 WT or S1PR2 R108A in WEHI-231 cells in response to the indicated amounts of S1P or FTY720-P treatment. Graphs show percent cells with detectable S1PR2 surface staining determined using flow cytometry. ( C ) Cell migration inhibition assay with S1PR2 WT WEHI-231 cells in response to S1P or FTY720-P at the indicated concentrations. Data are means ± SD (pooled from three independent experiments).

Article Snippet: The ligand S1P (Tocris) or FTY720 (S)-phosphate (FTY720-P, Echelon) was serially diluted in HBSS supplemented with 5 mM Hepes (pH 7.4) and 0.01% fatty acid–free bovine serum albumin (BSA; GoldBio) to prepare 10× stock solutions.

Techniques: Immunofluorescence, Staining, Flow Cytometry, Migration, Inhibition

Journal: Toxicology Research

Article Title: In vitro investigation of the toxicological mechanisms of Fingolimod (S)-phosphate in HEPG2 cells

doi: 10.1093/toxres/tfaf064

Figure Lengend Snippet: Effects of cytotoxicity observed with FTY720 (S)-P exposure on HepG2 cells after 24–48 and 72 h treatment. The cells were treated with 0.3125–10 μM concentrations.

Article Snippet: FTY720 (S)-P was purchased from Cayman Chemical Company (USA) and dissolved in dimethyl sulfoxide (DMSO).

Techniques:

Journal: Toxicology Research

Article Title: In vitro investigation of the toxicological mechanisms of Fingolimod (S)-phosphate in HEPG2 cells

doi: 10.1093/toxres/tfaf064

Figure Lengend Snippet: Effects of FTY720-S(P) (0.3125–10 μM) on cell viability by A) MTT, B) LDH and C) NRU assays in HepG2 cells after 72 h treatments. The cells were treated with 0.3125–10 μM concentrations. Data are expressed as mean ± SD, * P < 0.05 and * * P < 0.01 versus the control group.

Article Snippet: FTY720 (S)-P was purchased from Cayman Chemical Company (USA) and dissolved in dimethyl sulfoxide (DMSO).

Techniques: Control

Journal: Toxicology Research

Article Title: In vitro investigation of the toxicological mechanisms of Fingolimod (S)-phosphate in HEPG2 cells

doi: 10.1093/toxres/tfaf064

Figure Lengend Snippet: Changes in the ATP content observed with FTY720 (S)-P exposure on HepG2 cells after 72 h treatment. The cells were treated with 0.625–10 μM concentrations. Data are expressed as mean ± SD, * P < 0.05 versus the control group.

Article Snippet: FTY720 (S)-P was purchased from Cayman Chemical Company (USA) and dissolved in dimethyl sulfoxide (DMSO).

Techniques: Control

Journal: Toxicology Research

Article Title: In vitro investigation of the toxicological mechanisms of Fingolimod (S)-phosphate in HEPG2 cells

doi: 10.1093/toxres/tfaf064

Figure Lengend Snippet: Changes in mitochondrial membrane potential (MMP) observed with FTY720 (S)-P exposure on HepG2 cells after 72 h treatment. A) Quadrants of JC-1 aggregates (PE channel) and monomers (FITC channel), B) MMP levels. The cells were treated with 0.625–10 μM concentrations. Data are expressed as mean ± SD, * P < 0.05 and * * P < 0.01 versus the control group.

Article Snippet: FTY720 (S)-P was purchased from Cayman Chemical Company (USA) and dissolved in dimethyl sulfoxide (DMSO).

Techniques: Membrane, Control

Journal: Toxicology Research

Article Title: In vitro investigation of the toxicological mechanisms of Fingolimod (S)-phosphate in HEPG2 cells

doi: 10.1093/toxres/tfaf064

Figure Lengend Snippet: Oxidative stress production observed with FTY720 (S)-P exposure on HepG2 cells after 72 h treatment. A) Cellular ROS production, B) mitochondrial ROS production. The cells were treated with 0,625–10 μM concentrations. Data are expressed as mean ± SD, * P < 0.05, * * P < 0.01 versus the control group.

Article Snippet: FTY720 (S)-P was purchased from Cayman Chemical Company (USA) and dissolved in dimethyl sulfoxide (DMSO).

Techniques: Control

Journal: Toxicology Research

Article Title: In vitro investigation of the toxicological mechanisms of Fingolimod (S)-phosphate in HEPG2 cells

doi: 10.1093/toxres/tfaf064

Figure Lengend Snippet: Changes in antioxidant enzyme levels observed with FTY720 (S)-P exposure on HepG2 cells after 72 h treatment. A) Total superoxide dismutase (T-SOD), B) glutathione (GSH), (C) catalase (CAT). The cells were treated with 0.625–10 μM concentrations. Data are expressed as mean ± SD, * P < 0.05, * * P < 0.01 versus the control group.

Article Snippet: FTY720 (S)-P was purchased from Cayman Chemical Company (USA) and dissolved in dimethyl sulfoxide (DMSO).

Techniques: Control

Journal: Toxicology Research

Article Title: In vitro investigation of the toxicological mechanisms of Fingolimod (S)-phosphate in HEPG2 cells

doi: 10.1093/toxres/tfaf064

Figure Lengend Snippet: Molecular docking of FTY720 (S)-P depicted as 2D and 3D simulations within the binding sites of human SIRT3 (A) and SIRT5 (B), utilizing the X-ray crystallographic structures with PDB codes 4JSR and 5XHS, respectively.

Article Snippet: FTY720 (S)-P was purchased from Cayman Chemical Company (USA) and dissolved in dimethyl sulfoxide (DMSO).

Techniques: Binding Assay

Journal: Toxicology Research

Article Title: In vitro investigation of the toxicological mechanisms of Fingolimod (S)-phosphate in HEPG2 cells

doi: 10.1093/toxres/tfaf064

Figure Lengend Snippet: The Interaction profiles and docking scores of FTY720 (S)-P within the binding pocket of the SIRT3 (PDB ID: 4JSR) and SIRT5 (PDB ID: 5XHS) proteins.

Article Snippet: FTY720 (S)-P was purchased from Cayman Chemical Company (USA) and dissolved in dimethyl sulfoxide (DMSO).

Techniques: Binding Assay