Figure 3
PEITC restores the p53R175 mutant protein transactivational functions. (a) PEITC induced p53R175 mutant protein to bind chromatin. SK-BR-3 cells were treated with PEITC for 4 h and chromatin-bound and nuclear-soluble fractions were analyzed by immunoblotting. Histone H3 and Topoisomerase IIB served as markers for the chromatin and soluble nuclear fractions, respectively. (b) qRT-PCR of p53 regulated genes in SK-BR-3, H1299 and A549 cells treated with DMSO or 4 μM PEITC for 4 h. RNA was extracted and gene expression level was measured using TaqMan gene expression assay. (c) qRT-PCR of p53-regulated genes in NS siRNA or p53 siRNA-transfected SK-BR-3 cells treated with DMSO or 4 μM PEITC for 4 h. RNA was extracted and gene expression level was measured using TaqMan gene expression assay. (d) SK-BR-3, HOP92, AU565, H1299 and MEF ((10)3/175 and (10)3/273) cells were transfected with plasmid 16451 and were treated with PEITC (4 or 6 μM) for 24 h, respectively, followed by a luciferase reporter assay. (e) Western blotting analysis of p21 expression in SK-BR-3 cells treated with 4 μM PEITC or 20 μM etoposide for 4 h. A549 cell line was treated with 4 μM PEITC for 4 h as a control. Protein levels were determined by western blotting using p21, p53 DO-1 and GAPDH antibodies
