L165 041

asc-MDM, ascTAM, ascTU or CAF were serum-deprived for 24 h in serum-free OCMI basal medium prior to stimulation with PGI₂ analog MRE-269 (selexipag-active metabolite, Cayman Chemicals), Iloprost or Treprostinil (both Sigma Aldrich) for the indicated time points and concentrations. We have chosen these analogs due to different affinities to PTGIR and prostaglandin receptors, with MRE-269 as the most specific for PTGIR [26 (link)]. In individual experiments, cells were pretreated with 1 µM PTGIR antagonist CAY10449 or CAY10441 (both Cayman Chemicals) for 1 h before addition of PGI₂ analog. PPARβ/δ agonist L165041 (Biozol) was applied at 1 µM concentrations where indicated. For generation of CM, asc-MDM or ex vivo TAM were stimulated with PGI₂ analog under serum-free conditions for 0 or 24 h, 37 °C, 5% CO₂.

L165,041 was purchased from Biozol. ST247 was synthesized as described [20 (link), 57 (link)]. The inverse PPARβ/δ agonist PT-S264 is an optimized derivative of ST247 with improved plasma stability (Toth et al., manuscript submitted). Synthetic ligands were used at a concentration of 1 μM in all experiments unless indicated otherwise. Cells were treated for 24 h unless indicated otherwise. PUFAs were obtained from Biomol.