Figure 1.
AKT phosphorylation was induced under glucose deprivation.
(A) Immunoblotting analyses after incubation of HepG2 cells in the absence or presence of 5.5 mM of glucose and absence or presence of 30 µM of LY294002 for the indicated times. (B) HepG2 cells treated or not treated with various concentrations of glucose for 0.5 h were subjected to immunoblotting. (C) Immunoblotting analyses of HepG2 cells treated or not treated with 5.5 mM of glucose, 5.5 mM of galactose, or 5.5 mM of fructose for 0.5 h. (D) Immunoblotting analyses of HepG2 cells treated or not treated with 5.5 mM of glucose, 5.5 mM of 2-DG, or 5.5 mM of glucose plus 5.5 mM of 2-DG for 0.5 h.
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.
Figure 3.
SRC and OSSA are indispensable for the AKT phosphorylation induced by glucose deprivation.
(A) Immunoblotting analyses of HepG2 cells in the absence or presence of 5.5 mM of glucose in the and absence or presence of 20 µM of PP2 for the indicated times. (B) Addition of H2O2 to the culture medium containing 5.5 mM glucosein the absence or presence of 20 µM of PP2 for 0.5 h, followed by immunoblotting. (C) HepG2 cells were cultured in medium containing or not containing (glucose-deprived) 5.5 mM of glucose in the absence or presence of 30 µM of LY204002 or 20 µM of PP2 for 0.5 h. The cells were stained with 5 µM of BES-H2O2. ROS production was measured using flow cytometry. (D) siRNA-treated HepG2 cells were subjected to immunoblotting analyses using OSSA antibody. (E) Immunoblotting analyses of HepG2 cells transfected with a non-targeting siRNA or two separate OSSA siRNAs in the absence or presence of 5.5 mM of glucose for the indicated times. (F) Addition of H2O2 to the medium of OSSA-knockdown cells containing 5.5 mM glucose for 0.5 h, followed by immunoblotting.
Figure 4.
Induction of AKT phosphorylation under glucose deprivation is mediated by ROS generated by NOX4.
(A) siRNA-treated HepG2 cells were subjected to reverse-transcriptase PCR (RT-PCR) to confirm NOX4 knockdown. (B) NOX4 knockdown HepG2 Cells were stained with 5 µM of BES-H2O2 in the absence or presence of 5.5 mM of glucose for 0.5 h. ROS production was measured using flow cytometry. (C) Immunoblotting analyses of HepG2 cells transfected with a non-targeting siRNA or two separate NOX4 siRNAs in the absence or presence of 5.5 mM of glucose or treatment with exogenous H2O2 for 0.5 h.