Fig. 5. DSGOST dissociates between the Beclin-1 and Bcl-2 complex and activates AMPK/ULK1 pathway in gastric cancer cells.

a AGS and SNU-638 cells were treated with DSGOST (500 μg/mL) for 16 and 24 h. Bcl-2 was immunoprecipitated in AGS and SNU-638 cells, and the immunoprecipitated proteins were subjected to western blotting. Beclin-1 was detected in immunoprecipitates prepared with anti-Bcl-2 antibody by immunoprecipitation. Bcl-2 was also monitored in immunoprecipitates prepared with anti-Beclin-1 antibody by immunoprecipitation. b AGS and SNU-638 cells were treated with DSGOST (500 μg/mL) in a time-dependent manner (0, 8, 16, and 24 h). After DSGOST treatment, cell lysates were loaded to western blot assay and detected antibodies targeting p-AMPKα (Thr172), AMPKα, p-ULK1 (Ser757), ULK1, p-mTOR (Ser2448), mTOR, p-p70S6K (Thr389), and p70S6K for autophagy induction. β-actin was used as the protein loading control. c, d After AGS and SNU-638 cells were treated with DSGOST (500 μg/mL, 24 h) in the absence or presence of Compound C (Sigma, 2 µM), cell viability and LDH release were analyzed by WST-1 and LDH assays. Cell lysates were analyzed for the level of indicated proteins. e AGS cells were treated with DSGOST (500 μg/mL) for 16 and 24 h. AMPKα, mTOR, and ULK1 were immunoprecipitated in DSGOST-treated AGS cells and the immunoprecipitated proteins were subjected to western blotting using mTOR, AMPKα, and ULK1 antibodies, respectively. f, g Cell viability, LDH assay, and western blotting for LC3B, p62, and ULK1 were performed in gastric cancer cells treated with DSGOST (500 μg/mL) in the presence or absence of SBI-0206965 (Sigma, 10 µM) for 24 h. h, i After AGS and SNU-638 cells were treated with DSGOST (500 μg/mL, 24 h) in the absence or presence of control and ULK1 siRNA, cell viability and LDH assays were performed. Western blotting was carried out using antibodies for LC3B, p62, and ULK1. β-actin was used as the protein loading control. *p < 0.05