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PubMed Central, Figure 3: Nat Cell Biol. 2014 Oct; 16(10): 1004–13. Published online 2014 Sep 21. doi:  10.1038/ncb3040
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Nat Cell Biol. Author manuscript; available in PMC 2015 Apr 1.
Published in final edited form as:
Nat Cell Biol. 2014 Oct; 16(10): 1004–13.
Published online 2014 Sep 21. doi:  10.1038/ncb3040

Figure 3

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ΔNp63 but not TAp63 increases Wnt signaling via direct activation of Fzd7 expression

(a) GSEA of Wnt gene signatures in unsorted (upper panels) or P4 (lower panels) WT versus ΔNp63gfp/+ MECs. (b) Relative expression of Wnt luciferase reporter in P4 cells after lentiviral transduction of vector control, ΔNp63 or TAp63 lentiviruses (n = 3 samples; data represents mean ± s.d.). (c, d) Heat map of microarray data (c) and qRT-PCR (d) showing the expression of Wnt pathway genes in wild type and ΔNp63gfp/+ P4 cells from 7 week-old virgin mice. (e) qRT-PCR analysis of the Wnt signaling genes in control or p63-KD primary MECs. (f) qRT-PCR analysis of the Wnt signaling genes in control or ΔNp63 KD MECs. In d–f, n = 3 samples; data represents mean ± s.d. (g) Western blot analysis of ΔNp63 and Fzd7 in control, ΔNp63 and TAp63 KD MECs. (h) qRT-PCR analysis of the expression of FZD7 and WNT5A in control, ΔNp63, or TAp63 overexpressing HMLE cells at 48 h (n = 3 samples) or 10 days (n = 6 technical replicates pooled from 2 independent experiments) post-infection. Real time values were normalized to the housekeeping gene GAPDH. Data represents mean ± s.d. (i) ChIP-seq data from human keratinocytes show ΔNp63 binding region to a putative FZD7 enhancer. Dnase: DNAse I hypersensitive sites. Mamm cons: mammalian conserved areas. (j) ChIP analysis of p63 (using H129 and 4A4 antibody) binding to the FZD7 enhancer in HMEC (n = 3 samples; data represents mean ± s.d.). (k) ChIP analysis of p63 binding to Fzd7 enhancer in MMTV-Wnt1 primary mammary tumor cells (n = 6 technical repeats pooled from 2 independent experiments; data represents mean ± s.d.). (l, m) Relative expression of FZD7 enhancer-driven luciferase reporter in HMLE (l) and iMMEC (m) cells transiently transfected with indicated expression plasmids (n = 3 samples, data represents mean ± s.d.). *p < 0.05 by Student’s t test. Uncropped images of blots are shown in Supplementary Fig. 9.

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