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. 2013 Feb 1;18(4):386–399. doi: 10.1089/ars.2012.4615

FIG. 2.

FIG. 2.

p53 positively influences PGC-1α transcription. (A) SH-SY5Y cells were transfected with scramble (scr) or p53 siRNA [p53(−)] and treated with BSO for 15 h. Cells were lysed, and 20 μg of proteins was loaded for Western blot analysis of p53 and PGC-1α. Total RNA was isolated, and the relative mRNA level of PGC-1α was analyzed by RT-qPCR. Data are expressed as means±SD (n=6, *p<0.001 vs. BSO-untreated scr, °p<0.001 vs. BSO-treated scr). (B) Pifithrin (0.02 mM) was added 1 h before BSO treatment (15 h) and maintained throughout the experiment. Cells were lysed, and 20 μg of proteins was loaded for Western blot analysis of PGC-1α. (C) NCI-H1299 cells was transfected with pcDNA 3.1 empty vector (vector) or pcDNA 3.1 wild-type p53 cDNA [p53(+)]. L-NAME (0.01 mM) was added 1 h before BSO treatment (15 h) and maintained throughout the experiment. Cells were lysed, and 20 μg of proteins was loaded for Western blot analysis of eNOS and PGC-1α. (D) U0126 (260 nM) was added 1 h before BSO treatment (15 h) in SH-SY5Y cells and maintained throughout the experiment. Cells were lysed, and 20 μg of proteins was loaded for Western blot analysis of PGC-1α. (E) SH-SY5Y cells were treated with BSO treatment for the indicated times. Five hundred μg of nuclear protein extracts was subjected to oligo-pulldown assay by using the biotinylated oligonucleotide representing the putative p53RE on the PPARGC1A promoter and bound p53 was detected by Western blot. Twenty micrograms of nuclear proteins (input) was used for Western blot analysis of p53 and Sp1. (F) After 9 h from BSO treatment, ChIP assay was carried out on cross-linked nuclei from SH-SY5Y cells using p53 antibody followed by qPCR analysis of p53RE. Dashed line indicates the value of IgG control. Data are expressed as means±SD (n=3, *p<0.001 vs. control). (G) NCI-H1299 cells were transfected with pGL3 Vector containing the PPARGC1A promoter (+1/-1600 bp) together with pcDNA 3.1 empty vector (vector) or pcDNA 3.1 wild-type p53 cDNA [p53(+)]. Normalized luciferase activities were expressed as fol increase with respect to the values from control, which were set arbitrarily at 1. Data are expressed as means±SD (n=3, *p<0.001 vs. control). All the immunoblots reported are from one experiment representative of four that gave similar results. Actin was used as loading control. ChIP, chromatin immunoprecipitation assay.