5 iodoresiniferatoxin 1 rtx

Dulbecco’s modified Eagle’s medium, foetal calf serum and penicillin/streptomycin were from Corning (Corning, NY, USA). Bovine serum albumin (BSA) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Sphingosine-1-phosphate (S1P, cod.62570) and methanandamide (N-(2-hydroxy-1R-methylethyl)-5Z,8Z,11Z,14Z-eicosatetraenamide, cod.157182-49-5) were from Cayman Chemicals (Ann Arbor, MI, USA); N-arachidonoyl-ethanolamine (anandamide (AEA), cod. A0580) and 2-arachidonoil-glycerol (2-AG, cod. A8973) were from Sigma-Aldrich (St. Louis, MO, USA). AEA-d8 and 2-AG-d8 were purchased from Cayman Chemicals (Ann Arbor, MI, USA) and were used as internal standards. The selective antagonists of CB₁ SR141716A (SR1, cod. SML0800), of CB₂ SR144528 (SR2, cod. SML1899) and of GPR55 ML193 trifluoroacetate (ML193, cod. SML1340) were from Sigma-Aldrich (St. Louis, MO, USA). The TRPV1 selective antagonist 5′-iodoresiniferatoxin (I-RTX, cod. 1362) was from TOCRIS (Bristol, UK). For molecular biology studies RevertAid H Minus First Strand cDNA Synthesis Kit, from Thermo Scientific (Waltham, MA, USA), and SensiFASTTM SYBR Lo-ROX kit, from Bioline (London, UK) were used.

All reagents were purchased from Sigma-Aldrich (Milano, Italy) unless otherwise stated. Pentadecyl-2-oxazoline (PEA-OXA) and palmitoylethanolamide in an ultra-micronized formulation (referred to as PEA hereafter) were obtained from Epitech Group SpA (Saccolongo, Padova, Italy). Polyinosinic–polycytidylic acid (poly-(I:C)) was purchased from InvivoGen (Aurogene, Roma, Italy). 5′-Iodoresiniferatoxin (IRTX) and GW6471 were purchased from Tocris Bioscience (Space Import-Export, Milano, Italy). The deuterated standards—[²H]₈-AEA, [²H]₅-2-AG and [²H]₄-PEA—were purchased from Cayman Chemical (Cabru, Arcore, Italy). The human lung epithelial cell line (A549) was purchased from LGC Standards (Milano, Italy). The human IL-6 ELISA Kit and transforming growth factor beta (TGF-β) were purchased from Abcam (Prodotti Gianni, Milano, Italy). Total mRNA was isolated from A549 cells using Trizol Reagent (Thermo Fisher, Milano, Italy) following the manufacturer’s instructions. cDNA preparation from RNA was performed using iScript Reverse Transcription enzyme (Biorad, Milano, Italy). Specific primer sequences were designed using Primer3 Software (https://primer3.ut.ee/, accessed on 28 November 2022) and synthetized by Eurofin (Milano, Italy).

Sulfasalazine, 5-aminosalicylic acid (5-ASA, mesalamine), sulfapyridine, and capsaicin were purchased from Sigma (St. Louis, MO). 5′-Iodoresiniferatoxin (I-RTX) was purchased from Tocris Cookson (Ellisville, MO). Clostridium difficile toxin A was purchased from TechLab, Inc. (Blacksburg, VA).