Figure 2. ROS mediates AKT phosphorylation under glucose deprivation.

(A)(B)(D) HepG2 cells were cultured in either glucose-containing medium or glucose-deprived medium in the absence or presence of 12.5 mM of NAC for 0.5 h. ROS production was measured using flow cytometry. Cells were stained with (A) 5 µM of DCFDA or (B) 5 µM of BES-H₂O₂. Cells were gated within a range contained in the upper 5% of the total cell count under the glucose replete condition. (D) The AKT phosphorylation level was evaluated by immunoblotting. (C) Addition of H₂O₂ to media containing 5.5 mM of glucose in the absence or presence of 30 µM of LY294002 for 0.5 h, followed by immunoblotting.