Thle 3

The normal liver epithelial cell line THLE-3 and hepatocellular carcinoma cell line Hep3B, and Huh7 were obtained from ATCC (Manassas, VA, USA); Dulbecco’s Modified Eagle Medium (DMEM) and fetal calf serum, from Gibco (Grand Island, NY, USA); LipofectamineTM 2000 kit, TRIzol reagent, RNA reverse transcription kit, and PCR, from Invitrogen (Carlsbad, CA, USA); and PCR primers and bicinchoninic acid (BCA) protein detection kit, from Shanghai Shenggong Bioengineering Co., Ltd., (Shanghai, China). In addition, CCK-8 reagent was purchased from Sigma-Aldrich (St. Louis, MO, USA); crystal violet stain, from Zhongshan Golden Bridge Biotechnology Co., Ltd. (Beijing, China); Annexin V-Fluorescein Isothiocyanate (FITC)/Propidium Iodide (PI) Apoptosis Kit, from the Shanghai Meiji Biomedical Technology Co., Ltd. (Shanghai, China); Matrigel, from Sigma-Aldrich (St. Louis, MO, USA); and Transwell chamber; from the Shanghai Yanhui Biotechnology Co., Ltd., (Shanghai, China). E-cadherin, N-cadherin, and vimentin antibodies were acquired from the Santa Cruz Co., (Santa Cruz, CA, USA). pmiR-GLO luciferase reporter vector: Promega Corporation (Madison, WI, USA), dual-luciferase activity detection kits were obtained from the Shanghai Biyuntian Biotechnology Co., Ltd (Shanghai, China). Streptavidin magnetic beads were purchased from New England Biolabs (Beverly, MA, USA).

The following mammalian cell lines were purchased from ATCC through Cedarlane, Burlington, ON, Canada: MCF-7 (ATCC-HTB-22, human breast epithelial adenocarcinoma), HepG2 (ATCC-HB-8065, human hepatocellular carcinoma), HOS (ATCC-CRL-1543, Human Osteosarcoma), D-17 (ATCC-CCL-183, Canine osteosarcoma), DH-82 (ATCC-CRL-10389, canine histiocytosis), MCF10-A (ATCC-CRL-10317, human breast epithelial cell), AML-12 (ATCC-CRL-2254, mice liver epithelial cell), THLE-3 (ATCC-CRL-11233, human liver epithelial), and CnOb (cell applications-canine osteoblast). DH-82, HOS, and D-17 cells were cultured in EMEM (Sigma–Aldrich, Oakville, ON, Canada), MCF-7 and HepG2 in DMEM (Gibco), CnOb in CnOb basal medium supplemented with growth medium (cell applications), MCF-10A cultured in basal medium supplemented with growth medium, and THLE-3 in BEGM with supplements (Lonza Bioscience, Burlington, ON, Canada), as recommended. The cells were supplemented with 15% fetal bovine serum as required (Fisher Scientific, Ottawa, ON, Canada) and antibiotic (100 U/mL penicillin and 100 µg/mL streptomycin). The cells were maintained in a 5% CO₂ humidified incubator at 37°C (Fisher Scientific, Ottawa, ON, Canada). All experiments were performed after the second passage of the cells and repeated at least three times, independently.

HCC cells including HLF, Huh-7, SNU-449, HepG2 and LM3 were obtained from COBIOER (Nanjing, China). HLF, Huh-7 and LM3 cells were grown in DMEM. SNU-449 cells were grown in RPMI-1640 medium. HepG2 cells were grown in MEM. All mediums were obtained from Gibco (Grand Island, USA). Human liver epithelial cell (THLE-3) was obtained from ATCC (Manassas, VA, USA) and kept in BEGM (Lonza/Clonetics Corporation, Walkersville). Cells were left to grow at 37 °C under a humid environment with 5% CO₂.

Normal human liver cell line THLE-3 (RRID: CVCL_3804), HCC cell lines Hep3B (RRID: CVCL_0326), and HEK293T cells (RRID: CVCL_0063) were purchased from ATCC (Manassas, VA, USA). Li-7 (RRID: CVCL_3840), HuH-7 (RRID: CVCL_0336), SNU-182(RRID: CVCL_0090), and HuH-6 (RRID: CVCL_4381) cells were provided by Cell Bank/Stem Cell Bank, Chinese Academy of Sciences. MHCC97H (RRID: CVCL_4972) and BEL-7405 (RRID: CVCL_6569) cells were purchased from Fenghbio (Changsha, China). These cell lines are not listed as a commonly misidentified cell line by the ICLAC. In this study, THLE-3 and all HCC cells with less than 15 generations were used. The cells were grown in RPMI 1640 and DMEM containing 10% FBS (Gibco, Grand Island, NY, USA), respectively. All cells were cultured at 37 °C with 5% CO₂, and all experiments were performed with mycoplasma-free cells. To study the protein stability and degradation, cells were treated with 20 µg/mL of CHX or MG132 for 24 h. sh-NC, sh-SYVN1-1, sh-SYVN1-2, sh-β-catenin-1 or sh-β-catenin-2 were from GenePharma (Shanghai, China). The full-length of FoxO1 or SYVN1 was cloned into the pcDNA3.1 vector. SFB-SYVN1, SFB-FoxO1, Myc-FoxO1, and HA-Ubiquitin were constructed as previously described [23 (link)]. HCC cells were transfected using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA).

Human primary hepatocytes (HPH) were obtained from ScienCell (cat# 5200, Carlsbad, CA, USA) and maintained in hepatocyte medium (ScienCell, cat# 5201) containing 5% FBS according to the manufacturer’s instruction. Human hepatocytes (THLE-2 and THLE-3) were purchased from ATCC (cat# CRL-2706 and cat# CRL-11233, Manassas, VA, USA, respectively) and were cultured in BEGM SingleQuots medium (Lonza, cat# CC3170, Basel, Switzerland) containing 10% FBS. Breast cancer cell line JIMT1 cells were purchased from DSMZ (Braunschweig, Germany, cat# ACC 589) and cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% FBS. Another breast cancer cell line MDA-MB-231 cells were purchased from ATCC (cat# HTB-26) and cultured in RPMI-1640 containing 10% FBS. Atezolizumab, ipilimumab, nivolumab, and doxorubicin were purchased from the pharmacy at NIH (Bethesda, MD, USA). Necrostain-1 was purchased from MedChemExpress (cat# HY-15760, South Brunswick Township, NJ, USA). RIP3 inhibitor, GSK872, was purchased from Sigma-Aldrich (cat# 530389, St. Louis, MO, USA).

HCC cell lines HepG2, HuH7, Li7, SNU387, and SNU182 were kindly provided by the Stem Cell Bank, Chinese Academy of Sciences, and MHCC97H cells were obtained from Beyotime Company (Shanghai, China). The human liver cells THLE3 were obtained from ATCC. HepG2, HuH7, and MHCC97H cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. Li7, SNU387, and SNU182 cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% FBS and 1% penicillin/streptomycin. Then, THLE3 cells were cultured in Endothelial Cell Medium (ECM) supplemented with 5% FBS, 1% endothelial cell growth supplement (ECGS), and 1% penicillin/streptomycin. All cells were maintained in a humidified atmosphere containing 5% CO₂ at 37°C.

Liver immortalized cell lines THLE-2 and THLE-3, available from ATCC (Manassas, VA, USA), were allowed to culture in the BEGM (Lonza/Clonetics Corporation, Walkersville, MD, USA). The 10% fetal bovine serum and 1% penicillin-streptomycin both procured from Gibco (Grand Island, NY, USA) were used to supplement cell culture medium. An environment with 5% CO₂ at 37°C was maintained for culture of cells.