Cym5541

Protease activated receptor 1 (PAR-1)-activating peptide (PAR1-AP/SFLLRN-NH₂) was from Bachem (Bubendorf, Switzerland). Crosslinked collagen-related peptide (CRP-XL) from University of Cambridge (Cambridge, UK). Sphingosine-1-phosphate, FTY720, SEW2871, CYM5520, CYM5541, CYM50260, A971432, Ex 26, TY52156, JTE-013, CYM50358 hydrochloride, PF543 hydrochloride, N, N-dimethylsphingosine (DMS) were from Tocris (Bristol, UK). The D-erythro-DihydroSphingosine-1-phosphate (DHS1P) was from Sigma Aldrich (Poole, UK). FTY720-Phosphate was from Insight Biotechnology (Wembley, UK). Mouse anti-human PAC-1 conjugated to FITC and mouse anti-human CD62P conjugated to PE were from BD Biosciences (Oxford, UK). Fura-2 AM cell permeant calcium indicator and ActinGreen 488 ReadyProbes Reagent were from ThermoFisher Scientific (Loughborough, UK). Unless indicated, all other materials were from Sigma Aldrich (Poole, UK).

For treatment with S1PR modulators, Schwann cells were seeded at a density of 25,000–35,000 Schwann cells/cm² on PDL-coated 24-well plates. On day 2 after seeding, Schwann cells were incubated in a Schwann cell medium together with S1PR modulators for another 48 h. Fingolimod (0.1 µM FTY720P, Echelon Biosciences, Salt Lake City, UT, USA) was always used as a positive control. In addition, S1PR agonists were used to specifically modulate receptors S1P1 as well as S1P3-5. All substances were dissolved in DMSO (Sigma-Aldrich) unless otherwise stated: 7.5 µM Ponesimod (S1P1 agonist, Selleckchem, Houston, TX, USA); 15 µM EX26 (S1P1 antagonist, Tocris Bioscience, Bristol, UK); 5 µM CYM5541 (S1P3 agonist, Tocris); 5 µM TY52156 (S1P3 antagonist, Tocris), 1.25 µM, 2.5 µM, 5 µM and 10 µM CYM50260 (S1P4 agonist, Tocris); 1 µM CYM50358 hydrochloride (S1P4 antagonist, Tocris); 1.25 µM, 2.5 µM, 5 µM, and 10 µM A971432 (S1P5 agonist, Tocris, solved in 1N HCl). For each treatment condition, 3–4 wells of a 24-well plate were pooled and used for quantitative real-time-PCR (qRT-PCR) analysis.

Aspartic acid, D-[2,3-3H] was obtained from Perkin Elmer (NET581001MC, Boston, MA, USA). Pertussis toxin was acquired from List Biological Laboratories (#181, Campbell, Santa Clara Country, California, USA) and incomplete Freund’s adjuvant was obtained from Sigma-Aldrich (F5506, Saint Louis, MO, USA). Myelin oligodendrocyte glycoprotein (MOG) was purchased from Espikem (EPK1, Florence, Italy). Mycobacterium tuberculosis (H37Ra) was obtained from DIFCO BACTO Microbiology (241141, Becton, MD, USA). FTY720 was supplied by Novartis Pharma AG (Basel, Switzerland). N,N-dimethylsphingosine (DMS, 4640), sphingosine-1-phosphate (S1P, 1370), CS-2100 (4543, EC50 = 4.0 nM), CYM-5541 (4897, EC50 = 72–132 nM), Ex 26 (5833, IC50 = 0.93 nM), and TY-52156 (5328, Ki = 110 nM) were all purchased from Tocris Bioscience (Bristol, UK).

S1P was obtained from Cayman chemicals (BioNordika, Sweden). Selective agonists for S1PR1-5 were, respectively, 5-[4-Phenyl-5-(trifluoromethyl)thiophen-2-yl]-3-[3-(trifluoromethyl)phenyl]1,2,4-oxadiazole (≥ 99%, SEW2871 from Tocris Bio-Techne, Abingdon, UK), 1-[2-[2,5-Dimethyl-1-(phenylmethyl)-1H-pyrrol-3-yl]-2-oxoethyl]-1,6-dihydro-6-oxo-3-pyridinecarbonitrile (≥ 99%, CYM5520, Tocris), N,N-Dicyclohexyl-5-cyclopropyl-3-isoxazolecarboxamide (≥ 97%, CYM5541, Tocris), (2Z,5Z)-5-[[1-(2,4-Difluorophenyl)-2,5-dimethyl-1H-pyrrol-3-yl]methylene]-2-[(2-methoxyethyl)imino]-3-methyl-4-thiazolidinone (≥ 98%, CYM 50308, Tocris), 1-[[4-[(3,4-Dichlorophenyl)methoxy]phenyl]methyl]-3-azetidinecarboxylic acid (≥ 99%, A971432, Tocris). Fatty-acid free bovine serum albumin (BSA; Sigma-Aldrich) was used to prepare the vehicle for S1P delivery. Polyethyleneglycol of molecular weight 190–210 (PEG; Sigma-Aldrich #88440) was used as vehicle for S1PR agonists.