FIG. 4.

p53/PGC-1α/NFE2L2 axis induces SOD2 and γ-GCL. (A) After 15 h from BSO treatment, ChIP assay was carried out on cross-linked nuclei from SH-SY5Y cells by using NFE2L2 antibody followed by qPCR analysis of ARE sequence on the GCLC promoter. Dashed line indicates the value of IgG control. Data are expressed as means±SD (n=3, *p<0.001 vs. control; °p<0.01 vs. IgG). (B) SH-SY5Y cells were transfected with scramble (scr) or PGC-1α siRNA [PGC-1α(−)] and treated with BSO for 15 h. PGC-1α and NFE2L2 were detected by Western blot. (C) SH-SY5Y cells were transfected with scramble (scr) or PGC-1α siRNA [PGC-1α(−)] or p53 siRNA [p53(−)] and treated with BSO for 15 h. Nuclear protein extracts (500 μg) were subjected to oligo-pulldown assay using the biotinylated oligonucleotide representing the NFE2L2 consensus sequence on the GCLC promoter. (D) SH-SY5Y cells were transfected with scramble (scr) or PGC-1α siRNA [PGC-1α(−)] or p53 siRNA [p53(−)] and treated with BSO for 15 h. Cells were lysed, and 20 μg of proteins was used for Western blot analysis of SOD2 and γ-GCL. (E, F) SH-SY5Y cells were transfected with scramble (scr) or PGC-1α siRNA [PGC-1α(−)] or p53 siRNA [p53(−)] and treated with BSO for 15 h. Total RNA was isolated, and relative mRNA level of SOD2 (E) and NFE2L2 (F) was analyzed by RT-qPCR. Data are expressed as means±SD (n=4, *p<0.001 vs. BSO-untreated scr, °p<0.001 vs. BSO-treated scr). All the immunoblots reported are from one experiment representative of five that gave similar results. Actin or Sp1 were used as loading control.