Figure 2

Effect of IRS-1 overexpression on autophagy. (A) Cells were seeded and cultured for one day. Then, the amounts of LC3B-II between the IRS-1 overexpressing cells and the control cells were compared by Western blotting (left); the level of LC3B-II was normalized to that of actin for comparison (right). Autophagic vacuoles (black arrowheads) were observed under an electron microscope (Scale bar = 100 nm), and were counted in randomly selected groups of 30 cells. (B) Cells seeded and cultured for one day were further incubated in fresh DMEM containing 10 % FBS, or in EBSS, an amino acid deficient medium, for 6 h in the presence or absence of 100 nM bafilomycin-A. The influence of amino acid deprivation on autophagy was determined from LC3B-II levels by Western blotting (Left). The levels of LC3B-II in the absence of bafilomycin-A were normalized to that of actin for comparison (right). (C) Cells seeded and cultured for one day were further incubated in fresh serum free DMEM with or without 500 nM insulin for 6 h in the presence or absence of 100 nM bafilomycin-A. Using serum free DMEM avoided the confounding effects on autophagy of other growth factors that may be present in serum. The influence of insulin stimulation on autophagy was determined from LC3B-II levels using Western blotting (Left); the levels of LC3B-II in the absence of bafilomycin-A were normalized to that of actin for comparison (right). (D) Cells seeded and cultured for one day were further incubated in fresh DMEM containing 10 % FBS for 6 h in the presence or absence of 100 nM bafilomycin-A. LC3B-II levels were determined by Western blotting (Left), and the levels of LC3B-II in the absence of bafilomycin-A were normalized to that of actin for comparison (right). (E) Cells seeded and cultured for one day were further incubated in fresh DMEM containing 10 % FBS for the indicated time, and LC3B-II levels were examined by Western blotting.