Autophagy Inhibitor, 3-MA

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	5mM 3-MA can be used to treat the cells.

Protocol tips
5mM 3-MA can be used to treat the cells.

Publication protocol

Western blot analysis
After drug treatment for 24 h, QBC939 cells and HepG2 cells were harvested, washed with cold PBS, and incubated in RIPA lysis buffer for 40 min at 4°C to isolate total proteins. The concentration of total proteins was measured using the Bio-Rad assay. Protein samples (30 μg for each group) were separated by 12% SDS-PAGE and transferred to PVDF membranes (Roche, Basel, Switzerland), which were blocked with buffer (100 mM NaCl, 10 mM Tris–HCl [pH7.6], and 0.1% Tween 20) containing 5% non-fat dry milk for 1.5 h at room temperature. Membranes were incubated with various primary antibodies overnight at 4°C and with the corresponding secondary antibodies at room temperature for 1.5 h. The bands were visualized using Pierce ECL Western Blot Substrate (Thermo Scientific, Waltham, MA).

Flow cytometry
Cells were exposed to 20 mg/ml cisplatin and/or 50 μM CQ or 5 mM 3-MA for 8 or 12 h, trypsinized, and stained with Annexin V-FITC and propidium iodide (Annexin V Apoptosis Detection Kit, BestBio, Shanghai, China) to measure cellular apoptosis. Untreated cells were trypsinized and exposed to 50 μM 2-NBDG (Sigma-Aldrich) in glucose-free and serum-free DMEM for 30 min to evaluate the glucose uptake capacity. JC-1 (Beyotime Biotechnology, Shanghai, China) was used to evaluate MMP. Analysis was performed using a BD Accuri C6 flow cytometer (Becton Dickinson, Franklin Lakes, NJ).

Fluorescent staining
The production of total ROS and mtROS was evaluated by staining with DCFH-DA and MitoSOX, respectively. Cells were then incubated with 10 mM DCFH-DA for 30 min at 37°C or with 5 μM MitoSOX for 10 min at 37°C. After washing with PBS, the samples were observed using an IX71 fluorescence microscope (Olympus, Japan). Quantitation of the cell average fluorescence intensity was processed by software Image J (version 2.1.4.7), and more than 200 cells of each sample were analyzed.

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