IPEC-J2 Intestinal Cell Culture Protocol

The IPEC-J2 intestinal porcine enterocyte cell line (DSMZ, Braunschweig, Germany) was maintained, as previously described [11 (link)]. Moreover, the cells were cultured in high-glucose Dulbecco’s Modified Eagle’s Medium supplemented with 5% fetal bovine serum, 1% insulin–transferrin–selenium-X, and 1% (v/v) penicillin–streptomycin [55 (link)]. The cells were maintained at 37 °C in a humidified atmosphere of 5% CO₂. The IPEC-J2 cells were incubated with different treatments, including recombinant HBEGF (Mybiosource, San Diego, CA, USA), LY2944002 (Cell Signaling Technology, Danvers, MA, USA), SB202190 (Cell Signaling Technology, USA), and the AMPK inhibitor (dorsomorphin dihydrochloride; Santa Cruz Biotechnology, Dallas, TX, USA) at different intervals, as indicated in the figure legends.