Sc19220

LPS was from E. coli O55:B5 (Sigma). Synthetic oligodeoxynucleotides (ODNs) were from Invitrogen and were resuspended in endotoxin-free Tris-EDTA buffer. The sequences of ODNs for positive activation of TLR9 at the indicated concentrations are: murine-specific CpG ODN 5′-TCCATGACGTTCCTGACGTT-3′ (CpG-1826, also referred to as CpG if not specifically indicated by types), human-specific CpG ODN 2216 5′-ggGGGACGATCGTCgggggg-3′ (CpG-2216, lowercase letters represent phosphorothioate linkage; capitals, phosphodiester linkage 3′ of the base). The sequence for non-nCpG control ODN 1982: 5′-TCCAGGACTTCTCTCAGGTT-3′ (nCpG). To prevent TLR9 and CpG colocalization in endosomal vesicles, thereby inhibiting TLR9 activation by its agonist, we pretreated cells with the inhibitory iCpG ODN 2088: 5′-TCCTGGCGGGGAAGT-3′ (iCpG) at the indicated concentrations for 30 min prior to stimulation with agonists. IL-10 and anti-mouse IL-10 mAb were from R&D systems. Cycloheximide (CHX), SB202190, U0126, JNK inhibitor II, LY294002, AG490, prostaglandin E₂ (PGE₂), indomethacin (Indo), and NS398 were from Calbiochem. Ruxolitinib was obtained from Selleck Chemicals. EP receptor antagonists SC-19220, AH-6809, and AH-2348 were from Sigma. PhosphoPlus Stat3 (Tyr705) antibody kit, including anti-phospho-Stat3 Ab, anti-Stat3 Ab, and untreated and IFNα-treated HeLa cell extracts, was from Cell Signaling.

L-NAME, ACh, PE, the EP3 antagonist L798106, and the EP1 antagonist SC19220 were purchased from Sigma (St Louis, MO, USA). The COX-1 selective inhibitor FR122047, the IP antagonist CAY10441, the TP antagonist SQ29548, TP agonist U46619, PGI₂ and standard compounds of COX products were bought from Cayman Chemical (Ann Arbor, MI, USA). The selective COX-2 inhibitor rofecoxib was purchased from US Biological (Salem, MA, USA). L-NAME, PE, ACh, and FR122047 were dissolved in distilled water, while PGI₂ was dissolved in carbonate buffer (50 mM, pH 10.0). SQ29548, L798106, SC19220, CAY10441, and rofecoxib were dissolved in DMSO at 2,000-fold working concentration (the final concentration of DMSO was 0.05/100, v/v). The concentration of an inhibitor or antagonist used was based on previous reports, in which a selective inhibition of the effect of its intended target was considered to be achieved^{23 (link), 35 , 45 (link), 46 (link), 50 (link), 51 (link)}.
The composition of physiological salt solution (PSS; pH 7.4 with 95%O₂-5% CO₂) was as follows (in mM): NaCl 123, KCl 4.7, NaHCO₃ 15.5, KH₂PO₄ 1.2, MgCl₂ 1.2, CaCl₂ 1.25, and D-glucose 11.5. The 60 mM K⁺-PSS (K⁺) was prepared by replacing an equal molar of NaCl with KCl.

LTD₄, PGE₂ and murine COX-2 were purchased from Cayman Chemical (Ann Arbor, MI). Non-essential amino acids, FBS, BSA, Fura-2 acetoxymethylester (Fura-2 AM), U73122, dantrolene, Gö 6983, ketoprofen, LY 171883, MK 571, NS 398, SC560 and SC19220, PD98059, ethidium bromide and acridine orange were purchased from Sigma Chemical (St. Louis, MO). LY 255283 was purchased from Tocris Biosc. (Bristol, UK). Arachidonyl trifluoromethylketone (AACOCF₃) and bromoenol lactone (BEL) were acquired from Alexis Corp. (San Diego, CA). [Methyl-³H]thymidine (20 Ci/mmol) and [5,6,8,9,11,12,-14,15-³H] arachidonic acid ([³H]AA) (60-100 Ci/mmol) were from American Radiolabeled Chemicals Inc. (St. Louis, MO), and AH 23838 was kindly provided by Glaxo-Wellcome (Stevenage, UK).