Ah6809

Infected larvae were exposed to the pan-COX inhibitor indomethacin (Sigma-Aldrich) at 10 μM, COX1 inhibitor SC560 (Cayman Chemical) at 5 μM, COX2 inhibitor meloxicam (Cayman Chemical) at 15 μM, EP2 receptor antagonist AH6809 (Cayman Chemical) at 5 μM or EP4 receptor antagonist AH23848 (Cayman Chemical) at 10 μM. These drugs were previously used in zebrafish larvae [21 (link)–23 (link),27 (link),28 (link)]. The indomethacin concentration used was based on published results. For all other drugs, multiple concentrations were tested and the highest concentration of each drug that did not cause lethality or edema in uninfected larvae was used. 1000X stock solutions were made in DMSO and 0.1% DMSO was used as a vehicle control. After rinsing infected larvae, pre-mixed E3 with drug was added to dishes containing larvae. For survival and CFU assays, larvae were transferred to 96-well plates, one larva/well in 200 μL of drug/vehicle solution. For imaging experiments, larvae were transferred to 48-well plates with 500 μL of solution. All larvae were kept in the same drug solution for the entirety of the experiment unless otherwise noted.

AlCl₃·6H₂O (Sinopharm Chemical Reagent Co., Ltd., China) and maltol (Aladdin, USA) were of analytical grade. Maltol solution(60 mM) was prepared by adding 0.3784g maltol into 50 ml of autoclaved PBS and 0.1207g AlCl₃·6H₂O was added into 25 ml of autoclaved PBS as AlCl₃ solution(20 mM). Aluminum-maltolate solution (10 mM) was prepared by adding 25ml maltol solution and 25 ml AlCl₃ solution. After filtered through 0.22 mm millipore filter, 60 mM maltol solution and 10 mM aluminum-maltolate solution was stored at 4°C until used [24 (link), 78 (link)]. SC19220, AH6809 and L-161982 (Cayman, USA) were dissolved in the DMSO (Sigma, USA) to be 10 mM reserve liquid. 17-phenyl trinor Prostaglandin E2 ethyl amide and CAY10598 (Cayman, USA) were dissolved in ethyl alcohol to be 10 mM reserve liquid. Butaprost and Sulprostone (Cayman, USA) were dissolved in methyl acetate to be 10 mM reserve liquid [24 (link)].

Human pancreatic cancer cell lines MiaPaCa-2, BxPC-3, PANC-1, and Capan-1 were purchased from the European Collection of Cell Cultures (Salisbury, UK). Cells were maintained in DMEM (MiaPaCa-2) and RPMI1640 (BxPC-3, PANC-1 and Capan-1) supplemented with 10% FBS, 100 unit/mL penicillin G and 0.1 mg/mL streptomycin sulfate. Five-week-old male nude mice were purchased from Charles River Japan (Yokohama, Japan), which were housed in specific pathogen-free conditions. The experimental protocols were performed in accordance with the Care and Use of Laboratory Animals of the University of Tokushima School of Medicine and were approved by the Animal Care and Use Committee. Recombinant human IGF-1 was purchased from PeproTech (Rocky Hill, NJ) and human HB-EGF and IGF-2 were purchased from R&D systems (Minneapolis, MN). The EP2-selective antagonist AH6809 and EP4-selective antagonist GW627368X were purchased from Cayman Chemical (Ann Arbor, MI). The pseudo-substrate of PKC-θ was purchased from Merck Millipore (Billerica, MA).

Fetal bovine serum (FBS) (10%) was obtained from AmericanType Culture Collection (ATCC, Manassas, VA). RPMI-1640 medium was obtained from Gibco (Invitrogen Corporation, Carlsbad, CA). phorbol 12-myristate 13-acetate (PMA), NS398 were obtained from Sigma–Aldrich (St. Louis, MO). EP1, EP2, EP4, receptor antagonists (GW848687, AH6809, AH 23848) and recombinant PGE2 and PGD2 were purchased from Cayman chemicals. EP1 antagonist (L-798-106) was purchased from Tocris Bioscience. cAMP agaonist (forskolin) was purchased from Sigma-Aldrich. COX-2 and actin antibodies, non-targeting siRNA pool (NS siRNA) and siRNA targeting COX-2 were obtained from Santa Cruz Biotechnologies (Santa Cruz, CA).

Butaprost and AH-6809 (Catalog no: 13740 and 14050) were purchased from Cayman chemical. MG-132 (Catalog no: 474790) was from Calbiochem. Geneticin (G-418) was purchased from Invitrogen. The antibody for EP2 receptor (C-Term) was from Cayman chemical (Catalog no: 101750) and COX-2 was from Santa Cruz (Catalog no:1745). The antibodies for ZO-1, Occludin, Claudin-1, Claudin-2 & Claudin-4 were purchased from Zymed laboratories, Invitrogen (Catalog no: 617300; 711500; 519000; 516100 & 329400, respectively). IFN-γ was from R&D Systems. Twelve well transwell plates (Catalog no: 3460) were from Corning, Costar. All other chemical were purchased from Sigma-Aldrich or otherwise mentioned.