A 967079

The physiologic salt solution used was Krebs-Henseleit, which had the following
composition: 118.0 mmol/L NaCl, 4.7 mmol/L KCl, 2.5 mmol/L CaCl₂, 1.2
mmol/L MgSO₄, 25.0 mmol/L NaHCO₃, 1.2 mmol/L
KH₂PO₄, 10.0 mmol/L glucose). Solutions with high KCl
content involved addition of appropriate amounts of a 3-M KCl solution (in distilled
water) directly to the organ bath to achieve the desired concentration. For some
experiments, barium ions (Ba²⁺) substituted for Ca²⁺ in the
physiologic salt solution.
(±)-β-Citronellol (95% purity; Code C83201), ACh (PubChem ID 24891113), atropine (ID
24890401), 5-hydroxytryptamine (ID 24278124), L-N^G-nitroarginine methyl
ester (L-NAME; ID 24278011), tetraethylammonium (TEA; ID 24277874), sodium
orthovanadate (ID 24899708), capsazepine (ID 24277967), indomethacin (INDO; ID
24278173), A-967079 (CAS Number 1170613-55-4), HC-030031 (CAS Number 349085-38-7),
thapsigargin (ID 24278762) and verapamil (ID 24277881) were purchased from
Sigma-Aldrich (USA).
In general, stock solutions were prepared in distilled water and stored at -20°C.
β-Citronellol was dissolved directly in physiologic solution containing 2% Tween 80
and sonicated immediately before addition in the bath chamber. The maximum
concentration of the vehicle in the organ bath was 0.01% (v/v).
Salts (all of analytical grade) were purchased from Sigma-Aldrich or Merck
(Germany).

Formaldehyde was bought as a 37% solution (formaldehyde saturated solution, assured concentration 37–37.5 mass%, stabilized by methanol 8–12%, Carl Roth, Karlsruhe, Germany). Formaldehyde was prepared in ultrapure water and freshly diluted in extracellular solution (in mM: 140 NaCl, 5 KCl, 2 CaCl2, 2 MgCl2, 10 HEPES, 10 glucose, pH 7.4). To avoid confusion between % of formalin and % of formaldehyde in water, the concentration of formaldehyde in mM is used throughout the manuscript. Ionomycin, carbonyl cyanide m-chlorophenyl hydrazine (CCCP), A967079, BCTC, dantrolene and thapsigargin were obtained from Sigma-Aldrich, ryanodine and L-buthionine sulfoximine from Santa Cruz (Ann Arbor, MI).

Most reagents were purchased from Sigma–Aldrich or Tocris, including WS 12 (4-methoxyphenyl-5-methyl-2-1-methylethyl-cyclohexanecaboxamide), menthol, AMTB (N-3-aminopro-pyl-2-3-methylphenyl-methoxy-N-2-thienylmethylbenzamide) hydrochloride, allyl-isothiocyanate (AITC), cinnamaldehyde, carvacrol, eugenol and A967079 (4-fluorophenyl-2-methyl-1-pentene-3-one oxime). Stock solutions of agonists and antagonists were prepared in DMSO and were stored at–20°C (except for A967079, stored at 4°C).

Dulbecco’s modified Eagles’ medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco (Grand Island, NY, USA). Cardamonin and cinnamaldehyde were obtained from the National Institutes for Food and Drug Control (Beijing, China). GSK1016790A, Ruthenium Red, Amiodarone, A-967079, and 2APB were purchased from Sigma-Aldrich (Saint Louis, MO, USA). Other chemicals were also obtained from Sigma-Aldrich if not stated otherwise. Cell culture dishes and 96-well microplates were acquired from Greiner (Frickenhausen, Germany).

A549 alveolar epithelial cells (American Type Culture Collection, Manassas, VA, USA) were cultured in Ham’s F–12 K (Kaighn’s modification) medium with 10% heat-inactivated fetal bovine serum, 100 μg/ml streptomycin, 100 U/ml penicillin and 250 ng/ml amphotericin B (all from Gibco/Life Technologies, Carlsbad, CA, USA) at 37 ^◦C in 5% CO₂. A549 cells were seeded on 24-well plates and grown for 48 h before the experiments. During the experiments the cells were cultured with the following compounds or their combinations as indicated: TNF-α, IL-1β, IFN-γ, IL-4, IL-13 (all from R&D Systems Europe Ltd, Abingdon, UK), the Janus kinase (JAK) inhibitors bariticinib and tofacitinib, the STAT6 inhibitor AS1517499, the TRPA1 agonist allyl isothiocyanate (AITC) and the TRPA1 antagonists HC-030031 and A-967079 (all from Sigma Aldrich, St. Louis, MO, USA).

Similarly as with DSS feeding, 3-10 larvae were fed 24 h with indicated concentrations (100 µM or 500 µM) of stilbenoid compounds PS, PSMME, isorhapontin and astringin, or 200 µM of TrpA1 antagonists A-967079 (Sigma-Aldrich) and HC-030031 (Sigma-Aldrich) mixed in 1-2 ml fly food. For experiments with DSS treatment, larvae were moved from DSS-containing food to new tubes with stilbene/antagonist-containing food.