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).
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