Figure 4
Involvement of oxidative stress in iron-induced downregulation of EPO and HIFs expression in HepG2 cells. (a) Upper panels; representative figures of RhoNox-1 staining in HepG2 cells with or without SFO. Lower panel: quantitative analysis of fluorescence intensity measured by microplate reader. The values are expressed as means±s.d. **P<0.01 vs vehicle treatment; n=8 in each group. (b) Upper panels; representative figures of DCFH-DA staining in HepG2 cells with or without SFO. Lower panel: quantitative analysis of fluorescence intensity measured by microplate reader. The values are expressed as means±s.d. *P<0.05 vs vehicle treatment; n=16 in each group. (c) Effect of antioxidant agent on SFO-induced oxidative stress. Quantitative analysis of DCFH-DA fluorescence intensity measured by microplate reader. The values are expressed as means±s.d. **P<0.01; n=16 in each group. (d) Restorative effect of tempol on SFO-induced EPO mRNA downregulation in HepG2 cells. The values are expressed as means±s.d. *P<0.05, **P<0.01 vs vehicle treatment; n=8 in each group. (e) Tempol has no effect on SFO-induced HIF-2α mRNA downregulation in HepG2 cells. The values are expressed as means±s.d. **P<0.01 vs vehicle treatment; n=8 in each group. (f) Restorative effect of tempol on iron-induced downregulation of HIF-2α and HIF-1α expression in HepG2 cells. Upper panel: representative figures of HIF-2α, HIF-1α, and tubulin. Lower panel: semi-quantitative analysis of densitometory for HIF-2α and HIF-1α expression. The values are expressed as means±s.d. *P<0.05 vs vehicle treatment; n=6 in each group. (g) ChIP analysis of HIF-2α protein and the Epo HRE containing promoter interaction in HepG2 cells. Co-precipitated DNA fragments were detected by PCR using primers spanning the EPO HRE region. (h) SFO suppressed HRE-luciferase activity in HepG2 cells. The values are expressed as means±s.d. *P<0.05 vs vehicle treatment; n=6 in each group.
