2 methyl 2h pyrazole 3 carboxylic acid 2 methyl 4 o tolylazo phenyl amide ch223191

A72, a canine fibrosarcoma cell line, was maintained in Dulbecco’s modified Eagle’s minimal essential medium (DMEM) and incubated at 37 °C and 5% CO₂ [27 (link),28 (link)]. CCoV type II, strain S/378, kindly provided by Prof. C. Buonavoglia (University of Bari Aldo Moro, Italy) was utilized throughout the study. A72 cells were used both for virus stocks growth and for virus titration [27 (link)].
2-methyl-2H-pyrazole-3-carboxylic acid (2-methyl-4-o-tolylazo-phenyl)-amide (CH223191) (Sigma-Aldrich, St. Louis, MI, USA), a synthetic and specific AhR competitive antagonist [15 (link),29 (link)], was solubilized in DMSO (Sigma-Aldrich, St. Louis, MI, USA) and used at a concentration of 2, 5, 10 and 20 μM.
Monolayers of A72 cells were pretreated for 1 h at 37 °C with DMEM 10% FBS containing different concentrations of CH223191 (2, 5, 10 and 20 μM). Then, cells were infected or not with CCoV, at a multiplicity of infection (MOI) of 0.05 or 5. After 1 h of adsorption at 37 °C, cells were incubated and processed at 24 h post infection (p.i.). CCoV was in culture medium throughout the course of the experiment.

All compounds were obtained from commercial sources, solubilized in dimethyl sulfoxide (DMSO), and stored protected from light. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) was obtained from LGC Standard. [³H]TCDD diluted in ethanol was from Hartmann Analytic. 1-Hydroxyphenazine (1-HP) was purchased from TCI Europe N.V., 6-Formylindolo [3,2-b] carbazole (FICZ) and 2-Methyl-2H-pyrazole-3-carboxylic acid (2-methyl-4-o-tolylazo-phenyl)-amide (CH-223191) from Sigma–Aldrich. 1-HP, CH-223191 and FICZ were kept at RT, 4 °C or −20 °C, respectively.