Fig. 3

MECL induces non-apoptotic but autophagic cell death. a Cells were treated with 0–100 μg/mL MECL for 24 h. After treatment, the cells were collected and stained with annexin V and PI and then analyzed by flow cytometry. *p < 0.05. b Quantification of AVO-positive cells by flow cytometry. Cells were seeded in 60 mm dishes and treated with the indicated concentration of MECL. After 24 h, the cells were stained with 10 μM of AO, trypsinized, and analyzed. *p < 0.05. c Nuclear Hoechst staining. Cells were treated with the indicated concentrations of MECL, DMSO (control), or Quercetin 50 μM (positive control) for 24 h, then stained with Hoechst 33342 and observed under a fluorescent microscope. Arrows indicate the formation of apoptotic bodies. d Representative images for increased AVO-positive cells. Cells were seeded in 60 mm plates for 24 h and treated with the indicated concentrations of MECL for an additional 24 h. Arrows indicate the formation of AVO. e AGS cells were transfected with an expression construct for LC3 fused to green fluorescent protein (GFP-LC3) for 24 h. Cells were treated with or without MECL for 24 h and visualized under a fluorescent microscope. Arrows indicate the puncta of LC3. f AGS cells were treated with 0–200 μM phytol for 24 h. After treatment, stained with acridine orange dye and observed under a fluorescent microscope. Arrows indicate the formation of AVOs