Mhcc97h

Human HCC cell lines (MHCC97H, MHCC97L, and HCC‐LM3) were obtained from Liver Cancer Institute (Zhongshan Hospital, Fudan University).²¹ The cells were incubated in Dulbecco's modified Eagle's medium (DMEM; Gibco)—high glucose supplemented with 10% fetal bovine serum (FBS; Gibco) in a humidified atmosphere of 5% CO₂ at 37°C. Prior to treatment, the cells were grown to 80%‐90% confluence. Then, MHCC97H and HCC‐LM3 cells (2 × 10⁵ cells/mL) were treated with galangin (50 μmol/L, Sigma, Purity ≥ 95%) for 48 hours.

Human hepatocarcinoma cell lines (HepG2, HepB3, MHCC97H, MHCC97L, SK-Hep-1, Huh7, PLC) and human normal hepatocyte cell line (LO2) were purchased from the Institute of Cell Biology of the Chinese Academy of Sciences (Shanghai, China). Cells were cultured DMEM or RPMI-1640 containing 10% fetal bovine serum at a constant temperature of 37 °C in a humidified chamber with a constant atmosphere of 5% CO₂. For the validation of the role of SCAND3 and Myo1g methylation, we used demethylating agent 5-Aza-2′-Deoxycytidine (Sigma, St. Louis, MO, USA) in both the MHCC97H and SK-Hep-1 cell lines. The cells were treated with four doses (0.0, 2.5, 5.0, or 10.0 µM) of 5-AZC, and the medium containing 5-AZC was changed every 24 h. After 72 h of incubation, the DNA methylation levels were analyzed. Twenty pairs of fresh histopathologically verified HCC tissues and tumor-adjacent tissues were collected from Sun Yat-sen University Cancer Center. The clinicopathological data and information for the 20 HCC patients who underwent hepatectomies are reported in Table S1.

Human HCC cell lines (HepG2 and MHCC-97H) were purchased from American Type Culture Collection (ATCC; Rockville, MD, USA). Cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM; HyClone, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin G and streptomycin at 37 °C in a humidified atmosphere containing 5% CO₂.
HepG2 cells were transiently transfected with small interfering ribonucleic acid (siRNA) constructs specific for GBA to knockdown the expression of GBA. The siRNA targeting sequences are provided in Supplementary file-Section 2. Transient transfection of HepG2 and MHCC-97H cells was performed using polyethylenimine (408727, Sigma–Aldrich, Germany). ART (S24000, shyuanye, Shanghai, China; IC₅₀ of ART in HepG2 cells: 38.38 μM, IC₅₀ of ART in MHCC-97H cells: 171.4 mM; all P < 0.05, Supplementary Fig. 1b) was used at 0.25, 0.50 and 1.00 IC₅₀ in experiments to investigate its anti-HCC efficacy in vitro. ART was used at 0.50 IC₅₀ to investigate the molecular mechanism by which it suppresses HCC. LTI-291 (10 nM, S1024, Selleck, Shanghai, China) was used to induce GBA expression in HepG2 cells. Bafilomycin A1 (BAF; 10 nM, S1413, Selleck, Shanghai, China) was used to inhibit late-stage autophagy in HepG2 cells.