Hek293 cells

HEK293 cells (ATCC, #CRL-1573) were cultured in DMEM (Wako, #043-30085) supplemented with 10% fetal bovine serum (FBS). HepG2 cells (JCRB, #JCRB1054) and HepG2.2.15.7 cells^{12 (link)} were maintained on collagen-coated dishes with DMEM/F-12 GlutaMAX (Thermo, #10565018) supplemented with 10% FBS, 10 mM HEPES, and 5 μg/mL insulin. To generate HepG2-tet-GAL9 cells, HepG2-Tet-On Advanced cells (Takara Bio, #630932) were transduced with a retroviral vector encoding the GAL9 gene fused to a tetracycline-responsive element, and then selected with 1 μg/mL puromycin. PHHs were purchased from PhoenixBio (#PPC-P12). For transient knockdown, HepG2 cells were transfected with mixed gene-specific siRNAs (Qiagen, #SI03188409 and #SI04259066 for GAL9, #SI00057596 and #SI03089023 for p62, #SI00096229 and SI00096236 for RNF13, #SI00069265 and #SI02633946 for ATG5, #SI00708484 and #SI04354700 for viperin) or control siRNA (Qiagen, #1027281) by using Lipofectamine RNAiMAX Transfection Reagent (Thermo, #13778030). Alternatively, PHHs were transduced with lentiviral particles carrying gene-specific shRNA (Santa Cruz, #sc-35444-V for GAL9, #sc-78079-V for RNF13, #sc-29679-V for p62, #sc-41445-V for ATG5).

HEK293 cells (ATCC) were seeded in 6-well plates at a cell density of 8 × 10⁵ cells/well and cultured for 24 h. The cells were transfected with complex of vector DNA (the FLAG-tagged N188Q ghrelin receptor (control) or the indicated FLAG-tagged ghrelin receptor mutants) and FuGENE HD transfection reagent (Promega). The next day, transfected HEK293 cells were harvested in PBS containing 1 mM EDTA, and then incubated for 30 min on ice with 2.5 μg ml⁻¹ anti-FLAG antibody (Wako) and 5 μg ml⁻¹ Alexa Fluor 488–conjugated anti-mouse IgG goat polyclonal antibody (Thermo Fisher Scientific) in FACS buffer (PBS, 2% FBS, 0.05% NaN₃). Cell-surface expression levels were evaluated by flow cytometry on a Guava EasyCyte Plus Flow Cytometer. FLAG-positive cells were defined as cell populations with signals greater than the top 5% of MOCK cells.

SHSY-5Y cells and HEK293 cells were purchased from Riken Cell Bank (Tsukuba, Japan). Human umbilical vein endothelial cells (HUVECs) were obtained from American Type Culture Collection (ATCC, Manassas, Virginia). SHSY-5Y cells were maintained in Dulbecco’s modified Eagle’s medium/Ham’s F-12 medium (FUJIFILM Wako, Osaka, Japan). HEK293 cells were cultured in Dulbecco’s modified Eagle’s medium (FUJIFILM Wako, Osaka, Japan). The medium for SHSY-5Y and HEK293 cells were supplemented with 10% fetal bovine serum, penicillin (100 units/mL), and streptomycin (100 μg/mL). HUVECs were cultured as previously described [8 (link)]. Cell cultures were maintained in a humidified atmosphere containing 5% CO₂ at 37 °C in the dark. Cells were seeded in glass-bottomed culture dishes 1–2 days before imaging.