Snu 449

The human HCC cell lines (Huh7, Bel-7402, SMMC-7721, HepG2, and SNU449) and the liver cell line LO2 were purchased from ATCC and incubated at a humidified incubator (condition: 37°C, 5% CO₂). All the HCC cells were grown in DMEM medium (Gibco), except Bel-7402 and SNU49 cells (RPMI 1640; Gibco), which contained 10% FBS (Gibco) and 1% penicillin/streptomycin (Sigma).

Ewing’s sarcoma cell line A673, liver cancer cell lines HepG2 and SNU449 were purchased from ATCC, and were cultured for less than 6 months after resuscitation. HEK293T, tested for 8 STR loci and the amelogenin gene, and A673 were grown in Dulbecco’s Modified Eagle’s medium; HepG2 was grown in Eagle’s Minimum Essential Medium; SNU449 was grown in RPMI 1640, supplemented with 10% fetal bovine serum (FBS) and 2 mM glutamine. All the cell lines were incubated at 37°C with 5% CO₂.
For starvation treatment, cells were cultured in serum-free media after the attachment; for hypoxia treatment, cells were cultured under 1% oxygen level after the attachment.

HCC cell lines including Hep3B (ATCC HB-8064), HepG2 (ATCC HB-8065), Huh7 (JCRB0403), PLC (ATCC CRL-8024), Mahlavu [42 (link)], and SNU449 (ATCC CRL-2234) were obtained and used in this study. Cells 1 × 10⁴ were cultured in Dulbecco’s modified Eagle medium (DMEM) culture medium (Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS, Gibco^®, Thermo Fisher Scientific Inc., Waltham, MA, USA), 1% nonessential amino acid (Gibco^®), 1% penicillin–streptomycin (Gibco^®), and 1% L-glutamine (Gibco^®) in 100 mm culture dishes. Cells were kept in a humidified incubator at 37 °C with 5% CO₂ and subcultured every 3–5 days. Cells were harvested 48 h after subculture. Total RNA was extracted using TRIzol reagent (Thermo Fisher Scientific Inc.) according to the manufacturer’s protocol. NanoDrop Spectrophotometers (Thermo Fisher Scientific Inc.) were used to determine the concentration of total RNA, and purified RNA was stored in a −80 °C freezer for future use.

The human liver cell line LO2 and hepatocellular cell lines SNU398, SNU449, MHCC97H, Hep3B, PLC/PRF/5, and HepG2 were purchased from the ATCC (Manassas, VA, United States). PLC/PRF/5, Hep3B, MHCC97H, and HepG2 cell lines were cultured in complete Dulbecco’s modified Eagle’s medium (Gibco, United States), and SNU398 and SNU449 cells were cultured in RPMI1640 medium (Gibco, United States); all cells were supplemented with 10% fetal bovine serum (FBS) at 37°C in a humidified incubator with 5% CO₂ in the air.
HCC cell lines (Hep3B, HepG2, and SNU398) were transfected with siRNAs targeting ALG3 or control siRNA (HIPPOBIO, China) using Lipofectamine 2000 Reagent (Invitrogen, United States). The efficiency of the knockdown was confirmed using qRT-PCR analysis. The siRNA used were as follows: si-NC: 5′-UUC​UUC​GAA​GGU​GUC​ACG​UTT-3′, ALG3 siRNA#1: 5′-GGU​UUC​GUG​UAC​AUC​UUU​AUG-3′, and ALG3 siRNA#2: 5′-GGA​CCU​GAG​UCU​ACC​CUC​AGG-3’.

Hepatocellular carcinoma cell lines: PLC/PRF/5(PLC5) and SNU-449 were purchased from ATCC; and Huh7 was obtained from Japanese Collection of Research Bioresources (JCRB) Cell Bank. Cells were all cultured in DMEM (Gibco 12800-058) containing 10% fetal bovine serum, 100 units/mL penicillin, 100 μg/mL streptomycin, and Non-Essential Amino Acids Solution (Gibco 11140-050). Human Umbilical Vein Endothelial Cells (HUVEC) were purchased from ScienCell and cultured in Endothelial Cell Medium (ScienCell).

Human HCC cell lines MHCC-97H, HUH7, Bel-7402, Hep3B, HepG2 and SMMC-7721 were purchased from the cell bank of the Committee on Type Culture Collection of the Chinese Academy of Sciences (CTCC, Shanghai, China). THLE-2 and SNU449 were obtained from ATCC. MHCC-97H, SNU449, and MHCC-97L cell lines were maintained in RPMI 1640 medium (GIBCO, USA). HEK293T, HUH7, Bel-7402, Hep3B, HepG2 and SMMC-7721cells were cultured in DMEM (GIBCO, USA). Cell lines were maintained in culture supplemented with 10% FBS (GIBCO, USA) and 1% penicillin/streptomycin (Thermo) at 37°C with 5% CO₂ in a humidified incubator (Thermo).