Skf96365

Stimulant stock solutions were prepared according to
manufacturer’s instructions. For mucus measurement and tissue imaging
experiments 10 μM Cch (Sigma Aldrich) was provided basolaterally.
Stimulants were added to tissue culture medium for organoid imaging experiment
at the following concentrations: acetylcholine (20 μM; Sigma Aldrich),
Cch (20 μM; Sigma Aldrich), forskolin (100 nM; Cayman Chemical) and
nicotine (10 μM; Sigma Aldrich).
Inhibitor stock solutions were prepared according to
manufacturer’s instructions. For mucus measurement and tissue imaging
experiments 4-DAMP (100 nM; Cayman Chemical) was supplied basolaterally. For
organoid imaging experiments inhibitors were added to tissue culture medium and
incubated with cells for 10 minutes prior to stimulation with Cch. Inhibitors
were used at the following concentrations; atropine (50 μM; Sigma
Aldrich), physostigmine salicylate (10 μM; Sigma Aldrich), tetraisopropyl
pyrophosphoramide (10 μM; Sigma Aldrich), 4-DAMP (100 nM),
(−)-Xestospongin C (10 μM; Tocris), SKF 96365 (10 μM;
Tocris), carbenoxolone (50 μM; Sigma Aldrich), bumetanide (50μM;
Sigma Aldrich), clotrimazole (30 μM; Sigma Aldrich), Chromanol 293B (10
μM; Sigma Aldrich) and Ryanodine (100 nM; Tocris).

mTLQP-21, R21A mutant and the hTLQP-21 peptides were synthesized as previously described (Cero et al., 2014 (link)). SKF-96365 and KN-62 were purchased from Tocris while AnC0A4 was purchased from EMD-millipore.

NPS-2143 (catalog #: 3626) and SKF-96365 (catalog #: 1147) were purchased from Tocris Bioscience (Minneapolis, MN). Calcium chloride, monosodium phosphate, disodium phosphate, sodium oxalate, hydrogen peroxide solution, L-ornithine monohydrate (catalog #: O2375), Pyr6 (catalog #: SML1241), Pyr10 (catalog #: SML1243), and 2-aminoethoxydiphenyl borate (2-APB; catalog #: D9754) were purchased from Sigma-Aldrich (St Louis, MO). Dulbecco’s Modified Eagle Medium (DMEM), Fetal Bovine Serum (FBS; catalog #: 26140079), penicillin and streptomycin and Fura-2-acetoxymethylester (Fura-2-AM) were purchased from Invitrogen (Carlsbad, CA). All the chemicals used were analytical grade.