Autophagy and Aggrephagy Assays in HeLa and Huh7 Cells

HeLa and Huh7 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (D6429, Sigma-Aldrich, St. Louis, MI, USA) with 10% FBS (F7524, Sigma-Aldrich, St. Louis, MI, USA) and incubated in 5% CO₂ at 37 °C. HeLa cells stably expressing ULK1-EGFP, GFP-Atg5, GFP-WIPI, tf-LC3, and GFP-TFEB were described previously [6 (link)]. For performing LC3 flux assays, 125 nM bafilomycin A1 (023-11641, Wako, Osaka, Japan) was used. For the aggrephagy experiment, cells were treated with 5 μg/mL puromycin (160-23151, Lot PTL1732, Wako, Osaka, Japan) for 4 h and then washed with new medium. Torin-1 (475991, Calbiochem, Darmstadt, Germany) was used at a concentration of 250 nM. Rutin (30319-04, Nacalai tesque, Kyoto, Japan) and was dissolved in 42% ethanol. Quercetin (10005169, Cayman, MI, USA) was first dissolved in DMSO at a concentration of 210 mM and then dissolved with 42% ethanol.