Plk1 represses the p73-mediated transcriptional activation. A–C , H1299 cells (5 × 10 4 cells) were co-transfected with the constant amount of HA-p73α expression plasmid (25 ng), 100 ng of p53/p73-responsive luciferase reporter construct bearing p21 WAF1 ( A ), BAX ( B ), or MDM2 ( C ) promoter and 10 ng of Renilla luciferase reporter plasmid (pRL-TK) in the presence or absence of the increasing amounts of FLAG-Plk1 expression plasmid (50, 100, and 200 ng). To standardize the amounts of plasmid DNA per transfection, pcDNA3 was added to yield a total of 510 ng of plasmid. Forty-eight hours after transfection, cells were lysed, and their luciferase activities were measured. Data were normalized and presented as mean values ± S.D. of three independent experiments. D , RT-PCR analysis. H1299 cells were co-transfected with the constant amount of HA-p73α together with or without the increasing amounts of FLAG-Plk1 expression plasmid. Forty-eight hours after transfection, total RNA was prepared and analyzed for the expression levels of p21 WAF1 , BAX, and MDM2 by RT-PCR. Amplification of GAPDH serves as an internal control.