Penicillin streptomycin

Human PB NK cells were cultured on 6-well cell culture plates in Immunocult-XF T cell expansion medium (StemCell Technologies, Vancouver, BC, Canada) supplemented with 1× penicillin/streptomycin (VWR, Radnor, PA), 500 IU mL⁻¹ IL-2 (StemCell Technologies), 0.2 μL mL⁻¹ CD2/CD3/CD28 T cell activator (StemCell Technologies), and 10 ng mL⁻¹ IL-15 (StemCell Technologies). Culture medium was replaced every 3 days, and cells were kept at a concentration no less than 1 × 10⁶ cells mL⁻¹. Human PB NK cells were expanded for 14–21 days prior to cell experiments. PB NK cells were purchased from StemCell Technologies. Cells were purified via FACS sorting using the surface marker CD56. All cell donors were ≥90% pure. SHSY5Y-GFP cells were cultured in RPMI 1640 (VWR, Radnor, PA) supplemented with 10% FBS (VWR, Radnor, PA) and 1× penicillin/streptomycin in T75 vented cell culture flasks. Cells were split every 2–3 days using trypsin (0.25%) (VWR, Radnor, PA).

The human hepatocellular carcinoma (HepG2) cell line, which is a hepatocyte-derived cell line and has been widely used as an in vitro hepatocyte model (e.g., see refs 45 (link)–47 (link)), was a kind gift from Dr. Zaiqing Yang (Huazhong Agricultural University, College of Life Science and Technology). HepG2 cells were grown in RPMI-1640 (11875, Gibco) supplemented with 10% fetal bovine serum (1660516, Gibco) and 1% penicillin-streptomycin (15070, Invitrogen). To produce the Leu-deprived medium, RPMI-1640 without leucine, arginine, and lysine (R1780, Sigma-Aldrich) was supplemented with 200 mg/L (final concentration) arginine (0953, Amresco), 40 mg/L (final concentration) lysine (M234, Amresco), 10% fetal bovine serum and 1% penicillin-streptomycin. When cells were grown to approximately 80% confluence, twelve dishes of cells were randomly divided into two groups treated as follows: 1) for normal group (Ctrl), six dishes of HepG2 cells were cultured in fresh complete medium for 50 min; 2) for Leu deprivation group (-Leu), six dishes of HepG2 cells were cultured in Leu-deprived medium for 50 min. All the cell cultures were performed at 37 °C under 5% CO₂. For iTRAQ experiments, two independent biological replicates were performed to increase the statistical confidence.

MDA-MB-231 cells purchased from ATCC were cultured in Roswell Park Memorial Institute-1640 (RPMI) with L-glutamine (Lonza) and supplemented with 10% fetal bovine serum (Thermo Scientific) and penicillin/streptomycin (Amresco). Cells were grown at 37°C with 5% CO₂.