Mechanistic Insights into Autophagy Modulation

Transfections were done using Lipofectamine 3000 (Thermo Fisher, L3000008) for plasmid DNA. Doxorubicin (Sigma-Aldrich, D1515) was diluted in water to 5 mM and used at 1 μM for 48 h. Bafilomycin A₁ (BafA; Sigma-Aldrich, B1793) was diluted in DMSO to 200 μM and used at 100 nM for 4 h. Chloroquine (CQ; Sigma-Aldrich, C6628) was diluted in DMSO to 50 mM and used at 20 μM for 4 h. Rapamycin (Sigma-Aldrich, R0395) was diluted in ethanol to 2 mM and used at 200 nM for 4 h. Leu-Leu methyl ester hydrobromide (LLOMe; Sigma-Aldrich, L7393) was diluted in DMSO to 1 M and used from 0.001 to 1.4 mM for 6 h. Sorafenib tosylate (MedChemExpress, HY-10201A) was used from 5 to 14 μM for 24 h in vitro and 100 mg/kg in vivo daily. FITC-dextran (Sigma-Aldrich, FD40S) was directly diluted in the medium to 0.1 mg/mL. MTT (Thermo Fisher, M6494) was diluted in PBS (137 mM NaCl, 2.7 mM KCl, 12.5 mM Na₂HPO₄, 1.8 mM KH₂PO₄, pH 7.4) to 5 mg/mL. For high starvation, cells were washed twice with PBS and starved in low glucose DMEM lacking amino acids (USBiological, D9800-13) and serum. To overexpress the TFEB variant with pronounced nuclear localization characteristics [45 (link)] the plasmid pCIP-caTfeb (Addgene, 79,013; deposited by Reuben Shaw) was transfected into Huh-7 VTRNA1-1 KO cells.