3 methyladenine m9281

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).

The immortalized RPTC cell line was originally obtained from Dr. Ulrich Hopfer (Case Western Reserve University) and maintained in DMEM/F-12 medium supplemented with 10% fetal bovine serum and growth factors [47]. The retrovirus packaging cell line (293-Phoenix) was provided by Drs. Xiongjie Jin and Nahid F. Mivechi (Augusta University). The cells were cultured in DMEM medium with 10% fetal bovine serum. Primary antibodies: anti-LC3B (NB100-2220), anti-PINK1 (BC100-494) and anti-FUNDC1 (NBP1-81063) were from Novus Biologicals; anti-SQSTM1 (ab56416), anti-IMMT/MIC60 (ab110329), anti-COX4I1 (ab153709) and anti-PPIB (ab16045) were from Abcam; anti-TOMM20 (sc-11415), anti-PINK1 (sc-517353), anti-PRKN (sc-133167), anti-BNIP3L/NIX (sc-166332) and anti-HSPD1 (sc-13966) were from Santa Cruz Biotechnology; anti-CASP3 (9665) and anti-cleaved CASP3 (9664) were from Cell Signaling Technology; anti DNM1L (BD Biosciences, 611113); anti-ACTB (Sigma-Aldrich, A5316). Secondary antibodies for immunoblot analysis were from Jackson ImmunoResearch Laboratories. CCCP (C2759), chloroquine (C6628) and 3-methyladenine (M9281) were from Sigma-Aldrich. BECN1 peptide (Tat-BECN1, sequence: YGRKKRRQRRRGGTNVFNATFEIWHDGEFGT) and its control peptide (Tat-scrambled, sequence: YGRKKRRQRRRGGVGNDFFINHETTGFATEW) were synthesized by GenScript.

Aurantiamide acetate was isolated from the aerial parts of C. terniflora DC in our laboratory. Cisplatin was provided by QiLu Pharmaceutical (Jinan, China). Bafilomycin A1 (BafA1; BML-CM110-0100) and rapamycin (BML-A275-0005) were obtained from Alexis Biochemicals (Lausen, Switzerland). 3-methyladenine (M9281) and chloroquine (CQ; C6628) were bought from Sigma-Aldrich (Shanghai) Trading Co., Ltd, Shanghai, China. EYFP (enhanced yellow fluorescence protein)-Golgi plasmid was purchased from Clontech Laboratories, Inc., Mountain View, CA, USA. MitoTracker Red CMXRos and propidium iodide (PI) were from Invitrogen Life Technologies, CA, USA Carlsbad. Alexa Fluor 488 Phalloidin was obtained from Molecular Probes, Inc., Eugene, OR, USA. DMEM and heat-inactivated horse serum were obtained from GIBCO-BRL, New York, NY, USA. Foetal calf serum (FCS) was purchased from Hangzhou Tianhang Biological Technology Co., Ltd., Hangzhou, China. Anti-LC3 and anti-actin primary antibody was purchased from Cell Signaling Technology Shanghai Biological Reagents Co., Ltd., Shanghai, China, and Santa Cruz Biotechnology (Shanghai) Co., Ltd., China respectively.