4.3. Western Blot-Based Detection of Autophagy Levels

To determine basal levels of autophagic flux in AraC-Res versus parental cell lines, AML cells were plated in a 6W plate (1 × 10⁶ cells in 2 mL RPMI + 10% FCS) and treated with 50 μM CQ for 6 h at 37 °C. Subsequently, cells were harvested, washed with PBS, and lysed in 20 μL of lysis buffer (50 mM Tris, 2 mM EDTA, 2 mM EGTA, 150 mM NaCl, 0.1% SDS, 1% NP-40 substitute, 1 μM Na₃PO₄ containing protease inhibitor cocktail, SIGMAFAST™ Protease Inhibitor Tablets, S8820). To determine autophagy levels during AraC treatment, cells were plated in a 6W plate (1 × 10⁶ cells in 2 mL RPMI + 10% FCS) and treated with indicated concentrations of AraC (which gave ~20% reduction in cell viability after 72 h of incubation) and incubated for 24, 48, and 72 h at 37 °C. Subsequently, 50 μM CQ was added and incubated for 2 h after which cells were lysed as described above.

Before Western blot analysis, protein concentrations of the lysates were determined using Bradford protein assays (Pierce™ Coomassie (Bradford) Protein Assay Kit, #23200, Waltham, MA, USA). A total amount of 20-μg protein was loaded and separated using gel electrophoresis (10%, 15%, or 4–15% gradient polyacrylamide gels depending on the target protein) and blotted on PVDF blotting paper (Trans-Blot Turbo RTA Midi 0.2 µm PVDF Transfer Kit, #1704273, Biorad, Hercules, CA, USA). Proteins of interest were detected by incubating with primary antibodies (Santacruz: LAMP1 sc-20011 HRP, p62 sc-28359). Abcam beta-actin AC-15-HRP (Thermo scientific: LC3B from the LC3B Antibody Kit for Autophagy, Thermo scientific, {"type":"entrez-nucleotide","attrs":{"text":"L10382","term_id":"293264","term_text":"L10382"}}L10382, Waltham, MA, USA) at a dilution of 1:200 for Santacruz and a dilution of 1:1000 for other antibodies in 5% BSA for 16 h at 4 °C. If the primary antibody was not directly HRP-conjugated, staining was followed by the incubation with appropriate secondary HRP-conjugated antibodies at a dilution of 1:2000 (Swine anti-Rabbit-HRP p0217/rabbit anti-mouse-HRP p0260, Dako, Denmark) for 1 h at room temperature. Imaging was performed using a Bio-Rad ChemiDoc™ Imager. Densitometry analysis was performed using ImageJ (ImageJ 1.37c).