Wortmannin w3144

C57BL/6 mouse primary brain microvascular endothelial cells isolated from brain tissue of P2 pups were purchased from Cell Biologics (Chicago, IL, USA). Cells were cultured at 37 °C, in a humidified environment, in the presence of 5% CO₂. They were fed every 2 days with a complete mouse endothelial cell culture medium, on 60 mm diameter dishes or 12-well slides. Two days after confluence was reached, the cells were incubated for 24 h with serum-free medium. The cells were then treated with various pharmacological agents, including 50 mM ethanol, 100 μM acetaldehyde, lysosomal protease inhibitors 10 μg/ml pepstatin A (P-5318, Sigma-Aldrich, St Louis, MO, USA) and 10 μg/ml E64D (sc-201280 A, Santa Cruz Biotechnology), 200 nM rapamycin (R0395, Sigma-Aldrich), 100 nM Bafilomycin A1 (B-1793, Sigma-Aldrich), 10 mM 4-methylpyrazole (4-MP, M-1387, Sigma-Aldrich), 30 mM 3-methyladenine (M9281, Sigma-Aldrich), 75 nM wortmannin (W3144, Sigma-Aldrich), Krebs medium (145 mM NaCl, 4.75 mM KCl, 1.18 mM KH₂PO₄, 1.18 mM MgSO₄ hydrate, 25 mM NaHCO₃, 2.5 mM CaCl₂, pH 7.4) – this medium is commonly used for starvation condition, and ethanol (25 to 200 mM).

Human non‐small‐cell lung cancer cell lines (A549, NCI‐H1299) cells were purchased from the Cell Resource Center, Shanghai Institute of Biochemistry and Cell Biology at the Chinese Academy of Sciences. Cells were maintained at 37°C in a humidified air atmosphere containing 5% carbon dioxide in RPMI1640 supplemented with 10% FBS. Rapamycin (R0395), and wortmannin (W3144) were purchased from Sigma.