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. Author manuscript; available in PMC: 2012 Jun 25.
Published in final edited form as: Oncogene. 2009 Oct 5;29(3):411–420. doi: 10.1038/onc.2009.309

Figure 5.

Figure 5

Hairy/enhancer-of-split related with YRPW motif 1 (HEY1) positively regulates p53-dependent transactivation in a promoter-specific manner. (a) HEY1 greatly enhances p53-inducible gene 3 (PIG3)-luciferase reporter activity. U2OS cells were transfected with 100 ng of p21-Luc, BAX-Luc or PIG3-Luc in the presence or absence of 200 ng of expression vector for HEY1. (b) Deletion of the p53-binding site in PIG3 impairs the HEY1-positive effect. U2OS cells were transfected with 100 ng of PGL3, PIG3-Luc or PIG3 delF-Luc and 200 ng of expression vector for HEY1. (c) E6-mediated p53 degradation abolishes HEY1-positive effect on PIG3-luciferase reporter. U2OS cells were transfected with 100 ng PIG3-Luc and expression vectors for HEY1 (200 ng) or E6 (200 ng). (d) HEY1-positive effect on PIG3 promoter depends on the p53 status. HCT116 p53+/+ and p53−/− cells were transfected with 100 ng PIG3-Luc and expression vectors for HEY1 (200 ng) and p53 (5 ng) as indicated. (e) The polymorphic change L94M and mutations affecting the nuclear localization signal (NLS) impair HEY1-positive effect on PIG3. U2OS cells were transfected with 100 ng PIG3-Luc and 200 ng of transfection vectors for HEY1, the variant L94M or the triple-point mutant K51A/R53G/R51A. After transfection, the cells were washed and incubated for 24 h. Subsequently, cell lysates were assayed using a dual-luciferase reporter system. Normalized values are expressed relative to the activity of the reporter in the absence of HEY1 (a), relative to the activity of the empty pGL3-Luc reporter in the presence of HEY1 (b) or relative to the activity of PIG3 reporter in the absence of other vectors (c–e). The results shown represent the averages of results of at least two independent experiments assayed in duplicate + s.d.