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Fig. 3. Epithelial differentiation in p63–/– prostate. Scale bars: 50 µm. Goblet mucinous cells were detected in only p63–/– prostate (A,C). (C) The mucinous secretion stained for Alcian blue (pH 2.8). (D) Double staining for p63 and Alcian blue; one week after grafting the mucinous (Alcian blue-positive) cells were undetectable in p63–/– urogenital sinus (UGS). Double staining for androgen receptor (AR) and Alcian blue (E) in p63–/– UGS grafts; a small number of Alcian blue-positive cells was detected in epithelium of proximal ducts at 10 days after grafting (E, arrows). Expression of luminal cell markers was examined in p63+/+ (F,H,J,L) and p63–/– (G,I,K,M) prostates. AR (F,G), Nkx3.1 (Nkx, H,I), and secretory proteins, mouse dorsolateral prostate (mDLP) (J,K) and mouse ventral prostate (mVP) (L,M) were expressed in the luminal cells in p63+/+ and p63–/– prostates. mDLP-positive and mVP-positive cells were found among Alcian blue-positive cells (K,M). mDLP was co-expressed with mucin (K, red and black arrows). Secretory granules contained both mucin and mDLP in some mucinous cells (K, red arrows). Activity of Src was examined in p63+/+ and p63–/– prostates (N-Q). In the p63+/+ prostate, activation of Src was detected in neurons (P, red arrows) and a subset of basal cells (P, black arrows) but not in luminal cells (N,P). In p63–/– prostate, Src activity was detected mainly in Alcian blue-positive cells, but some non-mucinous luminal cells were also strongly positive for active Src (Q, white arrows). Downstream effecters in the Src-Ras-MAP kinase signal transduction pathway (MEK1/2 and ERK1/2) were also activated (phosphorylated) in neurons (not shown) and basal cells (R,S, arrows) in p63+/+ prostate and in (mucinous and non-mucinous) luminal cells in p63–/– prostate (T). Even though MAP kinase signaling was active, p63–/– prostate epithelium was proliferation-quiescent one month after grafting. Phosphorylation of histone H3 (pH3, U,V) was not detected in areas where phosphorylation of MEK was detected in p63+/+ (U) or p63–/– (V). Both p63+/+ and p63–/– contained rare neuroendocrine cells as assessed by expression of synaptophysin (W,X).