Sk hep 1 cells

Confocal microscopy was performed using a Zeiss LSM700 laser scanning confocal microscope. The SK-HEP-1 cells (ATCC) or 293 cells (Life technologies) were seeded on a 35-mm glass-bottom dish (D35-20-1-N, In Vitro Scientific) and transfected with the indicated plasmids for 48 h. 5 μM CnB-GFP or rhodamine-conjugated CnB was then added to the cells and incubated for 30 min, followed by three washes with PBS or acid-stripping buffer. The localization of the fluorescence was determined.

SK-Hep-1 cells were purchased from American Type Culture Collection (VA, USA), and Hep3B, Huh-7, HepG2, SNU-475, and SNU-449 cell lines were obtained from the Korean Cell Line Bank (Seoul, Korea). The cell lines were tested for mycoplasma contamination (MycoAlert^TM Mycoplasma Detection Kit, LONZA, Rockland, ME, USA). Lysates of normal human liver tissue were purchased from Novus (CO, USA). All culture media were obtained from Welgene (Gyeongbuk, Korea). Liver cancer cells were maintained at 37 °C in 5% CO₂ and cultured in Dulbecco’s Modified Eagle’s Medium or RPMI 1640 supplemented with 10% fetal bovine serum, 100 units/ml of penicillin, and 100 µg/ml streptomycin. Cells were exposed to 10 µM methionine medium for 24 h prior to incubation in Hank’s balanced salt solution (HBSS) for amino acid starvation and in glucose-null or high-glucose medium (as the control) for glucose starvation.

The HepG2 and SK-HEP-1 cells were obtained from American Type Culture Collection (Manassas, VA, USA) and grown in DMEM supplemented with 10% heat-inactivated FBS, penicillin (100 units/mL), and streptomycin sulfate (100 μg/mL) at 37 °C in an atmosphere of 5% CO₂.

Five HCC cell lines were used in subsequent experimental investigations. SK-Hep-1 cells were purchased from the American Type Culture Collection (Rockville, MD, USA). Huh-7 cells were a gift from Dr. Hui-Ling Chiou (Chung Shan Medical University, Taichung, Taiwan). HepG2, PLC/PRF/5, and HA22T/VGH cells were purchased from the Bioresource Collection and Research Center and the Food Industry Research and Development Institute (Hsinchu, Taiwan). The SK-Hep-1, HuH-7, and HA22T/VGH cells were cultured in Dulbecco's modified Eagle's medium (DMEM), whereas the PLC/PRF/5 and HepG2 cells were cultured in minimum essential medium. The PLC/PRF/5 and HepG2 cells were supplemented with 10% fetal bovine serum (Hyclone Laboratories, Logan, UT, USA), 100 μg/mL NEAA, and 100 mg/mL penicillin-streptomycin (Sigma Chemicals, St. Louis, MO, USA). These cell lines were maintained in a humidified atmosphere containing 5% CO2 at 37 °C.

Human NSCLC cell lines A549 (RRID: CVCL_0023) and H1299 cells (RRID: CVCL_0060), and human liver adenocarcinoma SK-Hep-1 cells (RRID: CVCL_0525) were purchased from American Type Culture Collection (ATCC). These cells were cultured in standard Dulbecco’s Modified Eagle Medium (DMEM with 25mM glucose) supplemented with 10% fetal bovine serum, 1% penicillin, and 50ug/ml streptomycin. All cells were grown in a humidified cell incubator with 5% CO₂ at 37°C.