Figure 4.

Iron overload inhibits NF-E2-related factor 2 (Nrf2) expression in PC12 cells after 48 hours of culture.

(A) Quantification of Nrf2 mRNA in PC12 cells in the different groups as shown by quantitative PCR. â-Actin mRNA served as an internal standard. The relative levels of Nrf2 mRNA were analyzed using the comparative threshold cycle method (2 ^-ÄÄCt).

(B) Western blot assay results showing Nrf2 protein expression in PC12 cells. Nrf2 protein level is expressed as the absorbance ratio to â-actin.

(C) Immunofluorescence microscopy on PC12 cells showing that Nrf2 localizes both in the cytoplasm and nucleus, and that it is primarily localized in the nucleus in the NGG and HGG. In the 400 μmol/L FAC group, the fluorescence intensity was reduced and labeling was dispersed. Deferoxamine promoted the expression of Nrf2 in the FAC + DFO group.

(A, B) Data are shown as mean ± SD from triplicate experiments. One-way analysis of variance was adopted for multiple-group comparison. Two-tailed Student's t-test was used for intergroup comparison. ^aP < 0.05, vs. HGG; ^bP < 0.05, vs. 25 μmol/L FAC group; ^cP < 0.05, vs. 100 μmol/L FAC group; ^dP < 0.05, vs. 400 μmol/L FAC group.

NGG: Normal glucose concentration group; HGG: high glucose concentration group; FAC25: 25 μmol/L FAC group; FAC100: 100 μmol/L FAC group; FAC400: 400 μmol/L FAC group; FAC + DFO: 400 μmol/L FAC + 200 μmol/L DFO group.