Figure 3

GO treatment increases intracellular ROS and induces autophagy. (A) Wild-type NIH/3T3 cells were treated with 10 mU/ml GO for various time intervals, and intracellular ROS levels were measured. (B) & (C) Control cells and cells overexpressing IRS-1 were treated with 5 or 10 mU/ml of GO for 6 h. LC3B-II levels were determined by Western blotting (left), and the levels of LC3B-II were normalized to that of actin for comparison (right) (upper two panels). Bafilomycin-A (100 nM) was added to determine the autophagic flux integrity (bottom panels). (D) Wild-type NIH/3T3 cells without (1,2) or with (3,4) treatment of 10 mU/ml GO for 24 h were examined under an electron microscope. Black arrowheads indicate autophagic vacuoles accumulated in cytoplasm.