Vpc23019

L-[3,4-³H]-glutamic acid was obtained from PerkinElmer. S1P and VPC23019 were obtained from Avanti Polar Lipids. FTY720-phosphate was obtained from Novartis Pharma, AG and Cayman Chemical. RP001, Y27632, and JTE013 were from Tocris Bioscience. BAF312 (Siponimod) was obtained from Novartis Pharma AG and Apexbio. Pertussis toxin (PTX) from Bordetella pertussis was obtained from List Biological Laboratories.

The synthetic Mus musculus sphingosine 1-phosphate receptor 5, mS1P5 (Eurofins genomics, Ebersberg, Germany), was inserted into the pEGFP-N3 vector (Clontech) using XhoI and BamHI restriction enzymes. Reagents were obtained as follows: VPC23019 from Avanti Polar Lipids (Alabaster, AL, USA), JTE-013 from Tocris (R&D Systems, Minneapolis, MN, USA); Hygromycin B, G418 and Z-VAD-FMK from Invitrogen; S1P and FTY-720P were purchased from Enzo Life Sciences (Farmingdale, NY, USA). BrdU was obtained from Thermo Fisher Scientific (Waltham, MA, USA) and Phalloidin from Interchim (Montluçon, France). Rabbit polyclonal antibodies against p44/42 mitogen-activated protein kinase (ERK), phospho-p44/42 mitogen-activated protein kinase (phosphoERK), Akt, phospho-Akt (Ser473), FAK, phospho-FAK (Tyr575/577) were purchased from Cell Signalling Technology (Beverly, MA, USA). Mouse monoclonal antibodies against β-tubulin were from Sigma and against BrdU were from Thermo Fisher Scientific. Control (LT1017) and anti-S1P (LT1002) antibodies, a gift of Dr Sabbadini, were used as previously described [16 (link),20 (link)].

Fingolimod (FTY720) HCl was purchased from Selleckchem (Houston, TX, USA) and anti-Iba1 antibody was purchased from FUJI FILM Wako Pure Chemicals (Japan). Anti-iNOS antibody was purchased from Santa Cruz Biotechnology, while anti-Mannose Receptor (CD206) antibodies were purchased from Abcam. Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204), p44/42 MAPK (Erk1/2), Phospho-MEK1/2 (Ser217/221), MEK1/2, Phospho-JNK (Thr183/Tyr185), JNK, Phospho-p38MAPK (Thr180/Tyr182), p38 MAPK, and Na/K-ATPase antibodies were purchased from Cell Signaling Technology. The S1PR1 and S1PR3 antagonist VPC 23019 was purchased from Avanti Polar Lipids (Alabaster, USA).

All materials for cell culture, alexafluor-488-phalloidin and SYBR green were from Invitrogen/Life Technologies Inc. (Burlington ON, Canada). HDL, acetylated LDL (AcLDL) and apoA1 were from Biomedical Technologies, Inc (Stoughton MA, USA). Corning Transwell inserts, cell culture plastic-ware and Camco Quik Stain II were from VWR International (Mississauga, ON Canada). W146 and VPC23019 were from Avanti Polar Lipids (Alabaster AL, USA). Wortmannin, LY294002, SB203580, PD98059, Go6976 and Ro31-8220 were from EMD Millipore Corp. (Billerica, MA, USA). FTY720, SEW2871 and Y-27632 were from Cedarlane Labs (Burlington, ON Canada). Rat tail collagen I and FITC-labeled anti-rabbit IgG were from BD Biosciences (Mississauga, ON, Canada). Reagents for RNA isolation and cDNA synthesis were from Qiagen Inc. (Toronto, ON, Canada). All other suppliers were as indicated. All other reagents, including primers for qRT-PCR were from Sigma-Aldrich Canada Co. (Oakville, ON, Canada).

Sphingosine 1-phosphate, and D-erythro (S1P) were purchased from Enzo Life Sciences (Plymouth Meeting, PA). C₁₇-Sphingosine 1-phosphate (C₁₇-S1P), lysophosphatidic acid (LPA), lysophosphatidylserine (LPS), lysophosphatidylinositol (LPI), and VPC23019 were purchased from Avanti Polar Lipids Inc (Alabaster, AL). Pertussis toxin (PTX) was obtained from Wako Pure Chemical Industries (Osaka, Japan).
S1P, LPA, LPS and LPI were dissolved in methanol. Just before use, the methanol was evaporated and the reagents were resolved in PBS containing 0.4% fatty acid-free BSA (Sigma-Aldrich Co., St. Louis, MO). C₁₇-S1P was dissolved in methanol. VPC23019 and PTX were dissolved in DMSO.