FIG. 7.

p53 positively regulates PGC-1α transcription in mice upon GSH depletion. (A) C57/BL6 mice were treated with BSO (20 mM) and/or L-NAME (4 mM) for 5 weeks in drinking water. Brains and skeletal muscles were homogenized, and GSH content was assayed by HPLC. Data are expressed as nmoles of GSH/mg of proteins and reported as means±SD (n=5, *p<0.001 vs. control). (B) Brains and skeletal muscles were homogenized, and 50 μg of proteins was used for Western blot analysis of p53, SOD2 and PGC-1α. Density of immunoreactive bands was calculated using the software Quantity one (Bio-Rad), and data are shown as a ratio of protein/actin. Data are expressed as means±SD (n=5, *p<0.01 vs. control, °p<0.01 vs. BSO-treated cells). (C) Total RNA was isolated, and relative mRNA level of PGC-1α and SOD2 were analyzed by RT-qPCR. Data are expressed as means±SD (n=4, *p<0.01 vs. control, °p<0.01 vs. BSO-treated cells). ChIP assay was carried out on cross-linked nuclei from the brain (D) and skeletal muscle (E) by using p53 antibody followed by qPCR analysis of p53REs on the ppargc1a promoter (−564, −954, and −2317). Dashed line indicates the value of IgG control (n=3, *p<0.001 vs. control; °p<0.01 vs. IgG). All the immunoblots reported are from one experiment representative of four that gave similar results. Actin was used as loading control.