Elevated expression of 14-3-3γ correlates with mutations in p53. A. mRNA expression in NSCLC tumors. RNA was extracted from frozen specimens of normal and tumor tissues and the quantity of 14-3-3γ determined using quantitative RT-PCR. Fold changes were determined by 2-ΔΔCt by comparing Ct values of 14-3-3γ with GAPDH. The box plot depicts 2-ΔΔCt values for 14-3-3γ expression. p-values are for comparison of the levels of 14-3-3γ mRNA in tumor specimens with normal tissue, which is calculated using ΔCt values. B. Analysis of 14-3-3γ expression by Western blotting in NSCLC tumors. A Western blot showing expression of 14-3-3γ protein in 8 representative NSCLC tumors and protein loading was confirmed by β-actin. C. Gene copy number. Genomic DNA was extracted from normal and tumor tissues and the relative quantity of 14-3-3γ DNA determined using quantitative PCR from equal quantities of DNA. ΔCt values for 14-3-3γ were normalized against PI3KR1 (p85alpha regulatory subunit of PI3K) since the latter was found to be unaltered in NSCLCs. The fold changes were calculated by comparing normal and tumor tissues using 2-ΔΔCt. Y-axis shows the fold change comparing normal and tumor tissues. D. 14-3-3γ expression and p53 mutations in NSCLC tumors. p53 mutational status was determined in the same lung tumors used in panels A and B using RT-PCR amplification of p53 mRNA followed by direct sequencing. Tumors were then segregated based on p53 mutation status, (wt-p53) or (mut-p53) and the 14-3-3γ mRNA expression determined as in panel A. p-values were determined by comparing normal tissue, tumors with wt-p53 and tumors with mut-p53.