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. 2012 Aug 1;122(9):3170–3183. doi: 10.1172/JCI63608

Figure 3. ZEB1 downregulates miR-34a levels through ΔNp63.

Figure 3

(A) Luciferase reporter assays on KP cells cotransfected with p53 or empty expression vectors and reporters containing promoters from Mdm2, 14-3-3σ, or consensus p53-binding sites (Generic). Results are expressed relative to empty vector transfectants, set at 1.0. (B) Q-PCR analysis of miR-34a in WT or Trp63-null (p63–/–) MEFs. (C) Q-PCR values relative to 344SQ cells (left) and 393P_ZEB1 cells (right), set at 1.0. (D and E) Q-PCR values relative to 393P_vector cells (D) and control siRNA-transfected 344SQ cells (E), set at 1.0. (F) Location of putative E-boxes in the ΔNp63 promoter are shown above ΔNp63 promoter luciferase assays on cotransfected 393P cells. (G) ΔNp63 promoter reporter constructs are shown above luciferase activities in cotransfected 393P cells, expressed relative to basal reporter activity (set at 1.0). (H) ΔNp63 promoter constructs are WT or lack 1 (Mut-a and Mut-b) or 2 (Mut-a/b) proximal E-boxes. Luciferase activities in cotransfected 393P cells are also shown, expressed relative to basal reporter activity (set at 1.0). (I) Luciferase reporter constructs that do (Mut) or do not (WT) contain a site-directed mutation in a p53-binding site. Luciferase activities in cotransfected 393P cells are also shown, expressed relative to basal reporter (GL3) activity (set at 1.0). (J) DNA amount at specific (p53/p63) and nonspecific (NS) sites of the miR-34a promoter precipitated from transiently transfected 393P cells by anti-Myc antibody, corrected for nonspecific binding activity by IgG precipitation and expressed as percent p63 bound. P values were determined by 2-tailed Student’s t test. Data are mean ± SD (n = 3).