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. 2016 Apr 20;7(21):31177–31190. doi: 10.18632/oncotarget.8870

Figure 5. ZNF224 regulates promoter activity of p21 and p53 via miR-663a.

Figure 5

A. the putative miR-663a binding sequences for the p53 and p21 3′ UTRs. Human p53 and p21 3′; UTR fragments containing either the wild type (WT) or mutant (MT) miR-663a binding sequences were cloned downstream of the luciferase reporter gene in the psiCHECK vector. B. MCF-7 cells were co-transfected with psiCHECK vector (200 ng) and Renilla vector (100 ng) in the presence or absence of miR-663a (20 nM), and in the presence or absence of miR-663a antagonist (40 nM). Luciferase activity was normalized to Renilla luciferase activity. Data represent the mean ± SEM of three independent experiments (*, vs. luc-p53 or luc-p21 #1 or luc-p21 #2/LNA-control (ctrl); **, vs. luc-p53 or luc-p21 #1 or luc-p21 #2/LNA-ctrl/miR-663a). C. MCF-7 cells were co-transfected with psiCHECK vector (200 ng) and Renilla vector (100 ng) in the presence or absence of FLAG-ZNF224. Luciferase activity was normalized to Renilla luciferase activity. The expression of FLAG-ZNF224 was confirmed using anti-FLAG antibody (inset). Tubulin was used as a loading control. Data represent the mean ± SEM of three independent experiments (**, vs. luc-p53 or luc-p21 #1 or luc-p21 #2/FLAG-ZNF224 (−)). D. MCF-7 cells were co-transfected with psiCHECK vector (200 ng) and Renilla vector (200 ng) in the presence or absence of FLAG-ZNF224, and in the presence or absence of miR-663a antagonist (40 nM). Luciferase activity was normalized to Renilla luciferase activity. The expression of FLAG-ZNF224 was confirmed using anti-FLAG antibody (inset). Tubulin was used as a loading control. Data represent the mean ± SEM of three independent experiments (*, vs. luc-p53 or luc-p21 #1 or luc-p21 #2/LNA-ctrl; **, vs. luc-p53 or luc-p21 #1 or luc-p21 #2/FLAG-ZNF224/LNA-ctrl). *P < 0.001, **P < 0.01.