Sk hep1

Human HCC cell lines MHCC-97H, SK-Hep1, and Huh7 were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). VPS35-konckout (KO) SK-Hep1 cells were generated by lentiCRISPR system as described previously.¹⁵ (link) Adenoviral recombinant AdVPS35 was generated using the AdEasy system. Green fluorescent protein-expressing analogous adenovirus (AdGFP) was applied as a control. Cells were maintained in DMEM (Hyclone) supplemented with 10% fetal bovine serum (FBS; Gibco, Rockville, MD, USA), 100 mg/ml of streptomycin, and 100 unit/ml of penicillin in a humidified incubator at 37 °C with 5% CO₂ and 95% air.

The human HCC cell lines HepG2, SK-HEP-1, and Huh-7 were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Hep3B and PLC/PRF/5 HCC cells were obtained from the American Type Culture Collection (Manassas, VA, USA). All cell lines were cultured in Dulbecco's Modified Eagle Medium (DMEM; Gibco BRL, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS, Gibco BRL), 100 U/mL penicillin, and 100 U/mL streptomycin, and incubated at 37 °C under an atmosphere containing 5% CO₂.
TNF-α was knowdown by lentiviruses containing shot hairpin RNA (shRNA): TNF-α shRNA, 5′-GTAGCCCATGTTGTAGCAA-3′. A scrambled shRNA lentivirus containing a non-targeting sequence (5′-TTCTCCGAACGTGTCACGT-3′, named LV-shNon) was used as a control. The shRNA sequence was synthesized by Shanghai GeneChem Co. Ltd. (Shanghai, China). shRNA were cloned into pLKO.1 (GV248) lentiviral vectors. Culture supernatants containing sh-RNA were added to HCC cells in the presence of polybrene. The cells were selected using 2 μg/mL puromycin after 24 h.

Cell lines L02, SMMC-7721, SK-Hep1, Huh7, PLC/PRF/5 and Hep3B were purchased from the cell bank of the Chinese Academy of Sciences (Shanghai, China). MHCC97H and MHCCLM3 cell lines were established and got from Fudan Liver Cancer Institute of Zhongshan Hospital (Shanghai, China). With the atmosphere of 37 °C and 5% CO₂, 10% fetal bovine serum (Invitrogen, USA) were mixed in Dulbecco’s modified Eagle’s medium (DMEM) (HyClone, USA) to culture the cells.

Human liver cancer cell lines (Huh‐7, SK‐Hep‐1, Hep 3B, and Hep G2), murine liver cancer cell line (Hepa 1‐6), and HEK‐293T cells were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The human umbilical vein endothelial cell (HUVEC) was purchased from the American Type Culture Collection (Manassas, VA, USA). The human immortalized liver cell line Hep Li5 was kindly donated by Professor Lanjuan Li (State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, Hangzhou, Zhejiang, China) [21 (link)]. The cancer‐associated fibroblasts (CAFs) were kindly donated by Zhentao Yang (the First Affiliated Hospital of Zhejiang University School of Medicine, Hangzhou, Zhejiang, China) [22 (link)]. All cells in this study were cultured in the recommended medium (Dulbecco's Modified Eagle Medium; Thermo Fisher Scientific, Waltham, MA, USA) containing 10% fetal bovine serum (Gibco, Waltham, MA, USA) and maintained at 37°C in a 5% CO₂ incubator. A MycAway Plus‐Color Mycoplasma Test Kit (YEASEN, Shanghai, China) was used to detect mycoplasma contamination.