Mem non essential amino acids

HepG2 cells (RCB1648) purchased from Riken Cell Bank (Tsukuba, Japan) were maintained in Minimum Essential Medium Eagle (MEM, FUJIFILM Wako) containing 10% fetal bovine serum (FBS, Thermo Fisher Scientific Inc., Waltham, MA, USA), 1% MEM nonessential amino acids (FUJIFILM Wako), 100 units/mL penicillin G, and 100 μg/mL streptomycin (FUJIFILM Wako) at 37 °C under a humidified atmosphere containing 5% CO₂. Each sample was prepared in DMSO to achieve a final concentration of 0.5%.

HBMECs were cultured in RPMI 1640 medium (Thermo Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS, Hyclone, Marlborough, MA, USA), 10% Nu-serum (BD Biosciences, Franklin Lakes, NJ, USA), 2 mM glutamine, 1% MEM nonessential amino acids (Wako, Osaka, Japan), 1 × MEM vitamin (Sigma, Irvine, UK), 100 U/mL penicillin, 100 μg/mL streptomycin, and 1 mM sodium pyruvate (Thermo Scientific). E. coli K1 strains were resuscitated from −80 °C, cultured overnight, and then subcultured in fresh BHI medium at a dilution of 1:100 until strains were grown to the exponential phase at an OD₆₀₀ of 0.6. Bacteria were collected by centrifugation and resuspended in RPMI 1640 medium containing 10% FBS. HBMECs infected at a multiplicity of infection (MOI) of 100 were incubated for 1.5 h in a cell incubator. The HBMECs were then washed with phosphate-buffered saline (PBS) to remove unbound bacteria and incubated with new medium containing gentamicin (100 mg/mL) for 1 h to kill extracellular E. coli. HBMECs were washed, lysed with 0.5% triton X-100, and plated on Luria broth (LB) agar plates for the determination of CFUs. A binding assay was performed similarly to the invasion assay except that the gentamicin treatment step was omitted. All experiments were conducted in duplicate and performed at least in triplicates.

HepG2 cells (RCB1648, Riken Cell Bank, Tsukuba, Japan) were maintained in Minimum Essential Medium Eagle (MEM, Sigma-Aldrich Co. LLC., St. Louis, MO, USA) containing 10% fetal bovine serum, 1% MEM non-essential amino acids (FUJIFILM Wako Pure Chemical Corporation, Tokyo, Japan), penicillin G (100 units/mL), and streptomycin (100 µg/mL) at 37 °C under 5% CO₂ atmosphere.

The human iPS cell line (TkDN4-M: 4M) was a kind gift from Dr. M. Ohtsu at The Institute of Medical Science, University of Tokyo. Undifferentiated 4M were maintained on mitomycin C (MMC; FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan)-treated SNL feeder cells in human iPS cell medium (DMEM/Ham's F12 (FUJIFILM Wako) supplemented with 20% Knockout Serum Replacement (KSR; GIBCO BRL, Palo Alto, CA, USA), 1x MEM nonessential amino acids (FUJIFILM Wako), 0.5x penicillin streptomycin (PS; FUJIFILM Wako), 55 μM 2-melcaptoethanol (2 ME Gibco) and 7.5 μg/ml basic fibroblast growth factor (FGF2; Peprotech, Rocky Hill, NJ, USA). For passage, 4M colonies were detached with CTK solution, dissociated into single cells, and seeded onto MMC-treated SNL feeder (ECACC, Salisbury, UK) in human iPS cell medium once a week.

The undifferentiated human lung cancer cell line Calu-6 (ATCC, Manassas, VA, USA) was used. Calu-6 cells were cultured in Eagle’s minimal essential (EMEM) medium (Fujifilm Wako Pure Chemical Industries, Osaka, Japan) supplemented with 10% FBS, MEM non-essential amino acids (Fujifilm Wako Pure Chemical Industries, Osaka, Japan), 1 mM sodium pyruvate (Fujifilm Wako Pure Chemical Industries), and 0.1% penicillin-streptomycin (Life Technology Co., New York, NY, USA) under 5% CO₂ at 37 °C.

DC2.4 cells, an immature DC cell line, were cultured in RPMI 1640 culture medium (Wako) supplemented with 10% heat-inactivated fetal bovine serum (Gibco), 0.05 mM 2-mercaptoethanol, 10 mM HEPES, 1 mM sodium pyruvate, 100 μM minimal essential medium (MEM) non-essential amino acids, 100 U/mL of penicillin, and 100 μg/mL of streptomycin (Wako). RAW 264.7 cells, a mouse macrophage cell line, were cultured in Dulbecco’s modified Eagle medium supplemented with 10% heat-inactivated fetal bovine serum, 100 μM MEM non-essential amino acids (Sigma–Aldrich), 100 U/mL of penicillin, and 100 μg/mL of streptomycin (Sigma–Aldrich). DC2.4 cells (5 × 10⁴ cells) and RAW 264.7 cells (5 × 10⁴ cells) were seeded in 96-well culture plates, respectively, and incubated to adhere overnight at 37°C in a humidified atmosphere of 5% CO₂. After the incubation, the cells were treated with 10 μg/mL LH2171 and incubated for a further 4 h. Then, DC2.4 cells were stimulated with LPS from Escherichia coli 055:H5 (Sigma–Aldrich) at a final concentration of 1 μg/mL, and RAW 264.7 cells were stimulated with LPS from E. coli 0111:B4 (Sigma–Aldrich) at a final concentration of 10 μg/mL for 20 h. In all experiments, the cells were grown to 80–90% confluence.