p44 Functions in Prostate Epithelial Cells Figure 3. The Cre-mediated deletion of the p44 gene in prostate epithelial cells. (A) A diagram illustrating the procedure to generate p44- (p442/2) epithelial cells from the mouse whole prostate. (B) Epithelial cells were stained for CK5 and CK18 as indicated. The nucleus was stained with Sytox green. (C) Western blot analysis of whole cell lysates (10 mg) made from LNCaP (lane 1) or prostate epithelial (lane 2) cells analyzed with anti- AR, -p63, -p44, or -actin antibody as indicated. doi:10.1371/journal.pone.0049173.g003 development and in the nucleus in the fully developed prostate gland (.45 days) [25]. To further characterize these findings, we studied p44 subcellular localization in dorsal lateral prostate (DLP) epithelial cells of mice up to 780 days old. Consistent with previous publications [5], this study found p44 (p44 shown in brown; nucleus shown in blue) localized to the cytoplasm of prostate epithelial cells in mice that were 10 and 17 days old (Fig. 1A). The p44 nuclear translocation was evident in prostate epithelial cells of mice at 30 days after birth, and there was very little cytoplasmic p44 by 60 days (Fig. 1A). Strong p44 nuclear signals remained in the adult prostate of mice up to 780 days old. However, we observed increased cytoplasmic p44 protein levels in epithelial cells of mice 780 days old, and the distinct p44 nuclear staining disappeared in the hyperplasia region (Fig. 1A, bottom right panel). Antibodies to cytoskeletal keratin proteins have been used to study prostate cell differentiation [27,28]. Those studies demon- strated that changes in the levels of differentiation are accompa- nied by a distinct transition in the expression profile of individual cytokeratins (CKs). Epithelial cells in the adult prostate can be characterized as basal cells (CK5++/CK182), luminal cells (CK52/CK18++), and intermediate cells (CK5+/CK18+). In murine prostate epithelium, p44 nuclear translocation is associated with increased expression of CK18 and decreased expression of CK5 [28]. Production and secretion of prostatic proteins are the main physiological functions of the prostate gland [4,28,29,30]. We collected prostate secretory proteins from mice at the ages of 17, 30, and 60 days. Amounts of prostate-secreted proteins were significantly higher in mice at the ages of 30 (16.360.9 mg/ml) and 60 (26.761.0 mg/ml) days than in mice at the age of 17 days (4.860.8 mg/ml). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis confirmed this observation (Fig. 1B). To further characterize prostate differentiation, we examined the expression of mRNAs of three prostate-secreted proteins (probasin, PSP94, and SBP) [31] in the mouse whole prostate gland at ages 10, 17, 30, 60, and 180 days. Low levels of all three mRNAs were noted at 10 and 17 days old, and a dramatic increase in these mRNAs was observed at 30 days and thereafter (Fig. 1C). Thus, we confirmed that p44 nuclear translocation is associated with the functional differentiation of the prostate epithelium. PLOS ONE | www.plosone.org 4 November 2012 | Volume 7 | Issue 11 | e49173