Figure 3
Metformin triggers ESCC cell autophagy. (a) Representative images of AO (upper) and MDC (middle) staining of ESCC cells following treatment with metformin at the indicated concentration for the indicated time. For AO staining (upper), red color intensity shows acidic vesicular organelles, representing autophagolysosomes. For MDC staining (middle), punctate fluorescence in the cytoplasm indicates formation of autophagic vacuoles. In the bottom panel, EC109 cells transfected with GFP-LC3 plasmid were treated with metformin for 24 and 48 h. Control cells showed a diffuse expression pattern of GFP-LC3, whereas metformin-treated cells displayed GFP-LC3-II a punctate pattern, indicating formation of autophagosomes. Magnification: × 400 for AO and MDC stainings, and × 1000 for GFP-LC3. (b) EC109 cells were treated with the indicated concentration of metformin for 48 h, and then cells were stained with AO. Cell autophagy was analyzed by quantification of acidic vesicular organelles (AVOs) with AO using flow cytometry. (c) Effects of metformin on autophagy-related proteins assayed by western blot at different metformin concentrations (left) and different times (right). β-Actin was probed as the loading control. (d) Transmission electron microscopy (TEM) images showing autophagic vacuoles (arrows) observed in metformin-treated EC109 cells for 48 h (right). Starved cells were used as a positive control for autophagy (middle). No or few autophagic vacuoles are observed in control cells (left panel). Representative data from one of three independent experiments are shown in (a–c)
