Figure 4.

Δ40p53 attenuates the transcriptional activity of FLp53. (a, b) H1299 cells were transiently co-transfected with 1 μg PSVL-p53 vector expressing p53 full-length (FLp53) and 1 μg SV-p53Δ40 vector expressing Δ40p53 (Δ40p53). As controls, cells were transfected with empty vector (Con), 1 μg of PSVL-p53 (FLp53) and 4 μg SV-p53Δ40 (Δ40p53) vectors alone. After 48 h, cells were lysed and RNA was extracted. The mRNA expression levels of the p53 target genes p21 (a) and BTG2 (b) were measured by quantitative real-time PCR using specific primers. (c–e) H1299 cells were transiently co-transfected with 5 μg FLp53 and Δ40p53 at serial dilutions, as indicated. As controls, cells were transfected with empty vector (Con) or 5 μg FLp53. Purified Δ40p53 was used as a positive control for the protein size. After 24 h, cells were lysed, and protein and RNA were extracted. (c) Western blots displaying FLp53 and Δ40p53 protein levels. GAPDH served as a loading control. As can be seen, the strong SV40 promoter of Δ40p53 led to high levels of protein expression in comparison to FLp53, consisting of the late SV40 promoter. Therefore, serial dilutions of Δ40p53 were co-transfected with FLp53, and the mRNA expression levels of p21 (d) and BTG2 (e) subsequently measured by quantitative real-time PCR, using specific primers. The results indicate that even low levels of Δ40p53 are sufficient to attenuate FLp53 function and reduce the expression levels of p21 and BTG2. Genes expression levels were normalized to GAPDH. Graphs represent an average of three independent experiments. Error bars represents S.E.