Fig. 2: The expression of Puf-A is transcriptionally suppressed by p53.

A RT-PCR and Western blot analyses of PUF-A (Puf-A) TP53 (p53), and CDKN1A (p21) expression in p53^+/+- and p53^−/−-HCT116 cells were performed. GAPDH and β-actin were used, separately, as internal controls. B RT-PCR and Western blot. Analyses of PUF-A (Puf-A), TP53 (p53), and CDKN1A (p21) expression in H1299 cells after transduction with control lentivirus (−), or low (+) and high (++) titers of p53-expressing lentivirus were performed. GAPDH andβ-actin were used, separately, as internal controls. C Western blot analysis of Puf-A in A549 cells transfected with control (shNC) or shp53-expressing vectors was performed. β-actin was used as an internal control. D The promoter activity of PUF-A in H1299 cells after transfection with control lentivirus (−), or low (+) and high (++) amounts of p53-expressing lentivirus was determined. *p < 0.05 (one-way ANOVA). Similarly, the promoter activity of PUF-A in p53^+/+- and p53^−/−-HCT116 cells after transfection with p53-expressing lentivirus (+) was determined. Mean ± SD values (n = 3) are shown. *p < 0.05 (two-tailed t test). E Two p53-response elements (p53RE-1 and p53RE-2) with the specific sequences are shown in the PUF-A gene. The pRE-1 and pRE-2 constructs drove by DNA fragment containing the p53-binding sites p53RE-1 (−720 to −700 nt) or p53RE-2 (+3497 to +3532 nt), respectively; the pRE-1m and pRE-2m constructs consisted of the region with mutations. These plasmids were transduced in H1299 and p53^+/+ HCT116 cells. Normalized activity of luciferase reporter with a control plasmid was shown. Mean ± SD was presented.