Doxycycline Induces Mitophagy and Suppresses Production of Interferon-ß in IPEC-J2 Cells

Xing, Yang; Liqi, Zhu; Jian, Lin; Qinghua, Yu; Qian, Yang

IPEC-J2 cell line (Guangzhou Jennio Biotech Co, Ltd., China), a non-transformed intestinal cell line originally derived from jejunal epithelia isolated from a neonatal, unsuckled piglet and maintained as a continuous culture (Rhoads et al., 1994) , were propagated in high-glucose DMEM (Life Technologies, Shanghai, China) containing 10% FBS (Life Technologies, Shanghai, China), 16 mM HEPES (Life Technologies, Shanghai, China) and 100 µg/ml penicillinstreptomycin (Life Technologies, Shanghai, China) under a 5% CO 2 atmosphere at 37 • C. Cells were seeded in plastic tissue culture flasks (25 cm 2 flasks, Corning, Shanghai, China) at a density of 2 × 10 5 /ml and passaged every 72-90 h for a maximum of 30 passages. Rabbit anti-LC3B was purchased from Beyotime Institute of Biotechnology (Haimen, China). Mouse anti-β-tubulin and HRP-conjugated secondary antibodies were purchased from Multisciences (Hangzhou, China). Chemical reagents used in this study were chloroquine (CQ) and Carbonyl cyanide 3-chlorophenylhydrazone (CCCP) purchased from Sigma-Aldrich. Rapamycin was purchased from Gene Operation (Michigan, USA). DOX and rotenone were purchased from Beyotime Institute of Biotechnology. Poly (I: C) was purchased from InvivoGen. Doxycycline is dissolved in deionized water at stock concentration of 20 mg/ml.