Topfluor cholesterol

Cells were treated with TopFluor cholesterol (5 μmol/L, 810255P, Sigma‐Aldrich) before lysosome isolation by density gradient centrifugation using a Lysosome Enrichment kit (89839, Thermo Fisher Scientific). The lysosome fractions were diluted with a reconstitution buffer (250 mmol/L sucrose, 1 mmol/L DTT, 1 mmol/L MgCl₂, 50 mmol/L KCl, and 20 mmol/L HEPES, pH 7.2). For mitochondria isolation, cells stably expressing 3× HA‐EGFP‐OMP25 (#83356, Addgene) were homogenized in ice‐cold PBS with homogenizer and spun down at 800 × g at 4°C for 10 min. The supernatant was spun down at 8,000 × g at 4°C for 10 min to collect the pellets. The pellets were then suspended with reconstitution buffer. Isolated mitochondria and lysosomes were incubated in 1 mg/mL cytosol of Huh7 cells, 1 mmol/L ATP, 1 mmol/L GTP, and an ATP‐regenerating system (30 mmol/L creatine phosphate, 0.05 mg/mL creatine kinase) at 37°C for 30 min. To isolate mitochondria from the reaction, pre‐washed anti‐HA magnetic beads were added and incubated for 5 min with rolling (60 rpm). The beads were then washed with PBS 3 times and eluted with 80% methanol extraction buffer. The eluates were subjected to microscopy or a microplate reader (SpectraMAX i3×, Molecular Devices, San Jose, CA, USA).