Cell Culture

LX-2 cells, immortalized human HSCs, were purchased from Merck Millipore (Billerica, MA, United States). In all experiments, the cells were subjected to no more than 15 cell passages. The cells were cultured in Dulbecco’s modified Eagle’s medium (Thermo Fisher Scientific, Waltham, MA, United States) containing 2% fetal bovine serum (Moregate Biotech, Bulimba, QLD, Australia), 100 U/mL of penicillin, and 100 μg/mL of streptomycin (Wako Pure Chemical Industries), and maintained at 37°C in a humidified atmosphere of 5% CO₂. The medium was changed every other day. Human embryonic kidney cells (HEK293, provided by the RIKEN BioResource Center, Tsukuba, Japan) or their derivatives, which were stably transfected with the human Toll-like receptor (TLR) 4a, MD2, and CD14 genes (293/hTLR4A-MD2-CD14; InvivoGen, San Diego, CA, United States), were cultured in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum, 100 U/mL of penicillin, and 100 μg/mL of streptomycin. 293/hTLR4A-MD2-CD14 cells were activated by lipopolysaccharide (LPS; Sigma-Aldrich, St. Louis, MO, United States) treatment.