FIGURE 3.

MEP50 regulation of p21^Cip1 promoter activity: a role for p53. A, MEP50 suppresses p21^Cip1 expression. KERn were electroporated with the indicated plasmid, and, after 24 h, extracts were prepared for quantitative RT-PCR detection of p21^Cip1 mRNA and immunoblot detection of p21^Cip1 and MEP50. EV, empty vector. B, opposing action of MEP50 and p53. A schematic of the human p21^Cip1 promoter shows the Sp1 and p53 transcription factor response elements. The numbers indicate the distance in nucleotides relative to the transcription start site. KERn were transfected with 0.5 μg of p21-2316, which encodes the full-length wild-type p21^Cip1 promoter, or the promoter harboring mutations at the p53-1 or p53-2 sites, linked to luciferase. After 24 h, extracts were prepared for the luciferase activity assay. The values are mean ± S.E. (n = 3). C–E, KERn were transfected with 0.5 μg of PG13-Luc (the p53 gene promoter linked to luciferase) or p21-2326 and the indicated micrograms of pcDNA3 or pcDNA3-FLAG-MEP50. 48 h post-transfection, cell extracts were prepared and assayed for promoter activity. The values are mean ± S.E. (n = 3). *, p < 0.005.