Research Paper Volume 8, Issue 12 pp 3450—3467

Figure 3. NO/PGC-1α-mediated antioxidant pathway is inhibited after p53C124S overexpression in p53-null NCI-H1299 cells. (A, B) NCI-H1299 cells were transfected with pcDNA3.1 vector containing cDNA for wild type p53 (Wt-p53), single p53 mutant in DBD (p53C124S) or with empty vector (Mock). Cells were lysed and 20 μg of proteins were loaded for Western blot analysis of p53, PGC-1α, NFE2L2 and SOD2. Tubulin was used as loading control. (C) L-NAME (100 μM) was added 1 h before BSO treatment (15 h) and maintained throughout the experiment. Total RNA was isolated and relative mRNA levels of PGC-1α, NFE2L2, SOD2 and GCLC were analyzed by RT-qPCR. Data are expressed as means ± S.D. (n=3; *p<0.05 vs untreated Wt-p53; °p<0.001 vs BSO-treated Wt-p53 cells). (D) Nuclear proteins (500 μg) were subject to S-NO derivatization with biotin. After Western blot the nitrocellulose was incubated with p53 antibody for detection of p53-SNO. Sp1 was used as loading control. The possible presence of cytoplasmic contaminants was tested by incubating nitrocellulose with rabbit anti-LDH. Numbers indicate the density of immunoreactive bands calculated using the Software Quantity one (Bio-Rad) and reported as the ratio of p53-SNO/Sp1. (E) Nuclear protein extracts (500 μg) were subjected to oligo-pull-down by using the biotinylated oligonucleotide representing the p53RE on the PPARGC1A promoter and bound p53 was detected by Western blot. Twenty μg of nuclear proteins (input) were used for Western blot analysis of Sp1. (F) ChIP assay was carried out on cross-linked nuclei from Wt-p53 and p53C124S NCI-H1299 cells using p53 antibody followed by qPCR analysis of p53RE. Dashed line indicates the value of IgG control. Data are expressed as means ± S.D. (n=4; *p<0.05 vs untreated Wt-p53; °p<0.05 vs BSO-treated Wt-p53 cells). All the immunoblots reported are from one experiment representative of four that gave similar results.