Uk356618

Canine melanoma cells (MCM-N1 cell line; 13-year-old male dog; chromosome number, 2n = 74) obtained from DS Pharma Biomedical Co., Ltd. (Osaka, Japan). The other canine melanoma cell lines (CMe1, CMMe2 and LMe) were kindly provided by Dr. Takayuki Nakagawa (Laboratory of Veterinary Surgery, Graduate School of Agricultural and Life Sciences, The University of Tokyo) [39 (link)–41 (link)]. The cells were cultured in 75-cm² culture flasks containing DMEM-LG (FUJIFILM Wako Chemical Corp., Osaka, Japan) supplemented with 10% FBS, 100 unit/mL penicillin and 100 μg/mL streptomycin, and maintained at 37°C in the environment of a humidified incubator with 5% CO₂ as described previously [42 (link), 43 (link)]. The cells were treated with recombinant canine IL-1β (Kingfisher Biotech, Inc., Saint Paul, MN, USA) for the indicated periods and concentrations. For inhibitor treatment, the cells were pretreated with 2-((aminocarbonyl)amino)-5-(4-fluorophenyl)-3-thiophenecarboxamide (TPCA-1, 10 μM for 1 h, MedChemExpress, Monmouth Junction, NJ) or UK356618 (50 nM for 2 h, MedChemExpress, Monmouth Junction, NJ), then the cells were treated with recombinant canine IL-1β.

IPEC-J2 were seeded into a 6-well plate in a growth medium, and treated with 200 μM si-DUOX2 alone or in combination with MMP3 inhibitor UK356618 (MedChemExpress, Monmouth Junction, NJ, USA) for 48 h. After removing the growth medium, cells were cultured in serum-free medium for 24 h, which was centrifuged at 3000× g and 4 °C for 10 min and collected as conditioned media. conditioned media was stored at −80 °C before use.