H69 cells, immortalized human normal cholangiocytes (a gift from Dr. G. J. Gores, Mayo Clinic, MN), were maintained as described36 (link), 37 (link). Our immortalized small and large cholangiocytes were also maintained as described7 (link), 11 (link), 14 (link), 38 (link). Stable transfected knockdown of SCT or SR in murine large cholangiocyte lines using short hairpin RNA (shRNA) plasmids were established in our laboratory and maintained as described7 (link), 11 (link). Human primary hepatocytes and culture media for these cells were purchased from ScienCell Research laboratories (Carlsbad, CA) and maintained according to the manufacturer’s instruction. Cells (3 × 105) were cultured in T175 flasks containing 30 mL of culture media with 1× phosphate buffered saline (PBS), LPS (50 ng/mL, Sigma-Aldrich, St Louis, MO), or SCT (100 nM, Bachem, Bubendorf, Switzerland) for 72 hours. Supplement fetal bovine serum (FBS) was depleted of EVs by ultracentrifugation at 120,000 × g for 18 hours (Type 50.2 Ti rotor, Beckman Coulter, Brea, CA)39 . All cells were cultured at 37 °C and 5% CO2.
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