Delipidated serum

To lower the cholesterol content in the cells, A1G and MMG cell lines were seeded at either 1 × 10⁴ cells/well in a 96-well plate (cytotoxicity) or 4 × 10⁵ cells/well (cholesterol content) in a 6-well plate in complete Ham’s F-12 medium for 8 h to allow the cells to attach to the plate. Cells were then washed three times with Ham’s F-12 medium supplemented with delipidated serum (Biowest) (ΔFBS medium) and incubated with 10 µM zaragozic acid (ZA) in ΔFBS medium for 16 h at 37 °C, 5% CO₂. To reload the plasma membrane with cholesterol, cells after ZA treatment were washed three times with ΔFBS medium and incubated with 100 µM cholesterol–1 mM methyl-β-cyclodextrin complex [31 (link)] in ΔFBS medium for 30 min at 37 °C, 5% CO₂. As a control, cells were incubated for 16 h with only ΔFBS medium at 37 °C, 5% CO₂. After treatment, cells were either processed with AmB cytotoxicity MTT assay or lysed as described for cholesterol content measurement.

Sodium acetate (Sigma-Aldrich, S2889), C646 (Sigma-Aldrich, SML0002), orlistat (Cayman, 10005426), ACSS2 inhibitor (Millipore, 533756), mithramycin A (Cayman, 11434), pentamidine (Cayman, 20679), ¹⁴C₂-acetic acid, (Perkin Elmer, NEC553050UC), 2-deoxyglucose (Sigma-Aldrich, D8375), 2,4-dinitrophenol (Sigma-Aldrich, D198501), rotenone (Sigma-Aldrich, cat. no. R8875), DAPI (Invitrogen, P36935), Turbofect transfection reagent (Thermo Fisher, R0532), DharmaFECT 1 transfection reagent (Horizon Discovery, T-2001), Nile red (Thermo Fisher, N1142), protein G magnetic beads (Thermo Fisher, 10004D), dispase II (Sigma-Aldrich, D4693), delipidated serum (Biowest, S181L), TRIzol (Thermo Fisher, 15596026), MG132 (EMD Millipore, 474790), PowerUP SYBR Green Mastermix (Applied Biosystems, A25742), MTT (Sigma-Aldrich, M2128), HA-magnetic beads (Thermo Fisher, 88836).