p44 Functions in Prostate Epithelial Cells Figure 9. Cytoplasmic p44 promotes and nuclear p44 inhibits proliferation of prostate epithelial cells. (A) Nuclear p44 enhanced expression of prostate-secreted proteins. A, Quantitative RT-PCR analysis of expression of prostate-secreted proteins. Total RNAs were isolated from LTts-ECs cultured at 33uC or 37uC for 6 days, and amounts of specific RNAs were measured by RT-PCR. Relative expression indicates specific AR expression in LTAts-ECs cultured at 37uC divided by amounts of the same RNA in LTAts-ECs cultured at 33uC. (B) Cytoplasmic p44 fully restored the growth inhibition of prostate epithelial cells induced by the non-permissive temperature. Growth curves of LTAts-ECs infected with control lentivirus (Og2) or lentivirus expressing p44, NES-p44, or NLS-p44 and grown at 33uC or 37uC. One-way ANOVA analysis: F = 196.5, p = ,0.0001, R square = 0.9861. The p value of unpaired Student’s t-Test of data between 37uC-Og2 and 33uC-NESp44, 33uC-NESp44, 37uC-NESp44, 33uC-NLSp44, or 37uC-NLSp44 is ,0.0001, ,0.0001, 0.0053, 0.0017, or 0.0038. (C) Nuclear p44 inhibited proliferation of prostate epithelial cells. LTAts-ECs infected with control lentivirus (Og2) or lentivirus expressing p44, NES-p44, or NLS-p44 were allowed to grow in the presence of BrdU and immunostained with an anti-BrdU antibody (shown blown). doi:10.1371/journal.pone.0049173.g009 Materials and Methods Animals Mice were handled in accordance with the guidelines published in the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The MD Anderson Institutional Animal Care and Use Committee approved all the experimental procedures used for mice. Mice were sacrificed by CO2. Mouse Prostate Preparation Male C57BL6/J mice (n = 40) at the age of 7, 14, 30, 60, 180, 360, 510, or 780 days were injected with BrdU (0.1 mg per g of body weight per day; Sigma-Aldrich, St. Louis, MO) via intraperitoneal injection for 3 days. Mice were killed, and whole prostate glands were freed from surrounding structures and fixed by immersion in 4% paraformaldehyde in phosphate-buffered saline (PBS) overnight at 4uC. The tissues were then embedded in paraffin, and sections (4 mm) were cut and mounted on Super-frost Plus adhesion slides (Fisher, Pittsburgh, PA) for hematoxylin and eosin (H&E) and immunohistochemical staining. Collection of Secretions Luminal material was collected from mouse prostate glands. The whole prostate was minced in 0.1 ml of PBS containing protease inhibitors (1 mM phenylmethane sulfonylfluoride, 10 mM leupeptin, 1.4 mM pepstatin A). The tissue was then centrifuged at 16,0006g for 2 min at 4uC, the supernatant was drawn off, and the protein concentrations were determined by using the Bradford method (Bio-Rad) with bovine serum albumin (Bio- Rad) as the standard. The supernatant was mixed with 0.1 ml of 26SDS gel sample buffer. The samples were then heated to 100oC for 5 min and on a 5 to 20% SDS polyacrylamide gradient gel. The gel was stained with Coomassie blue R 250. Isolation and Culture of MPECs Male p44loxP/loxP (n = 5) or H-2Kb-tsA58 (n = 5) mice were killed at the age of 21 days. The whole prostates were then microdissected, minced, and incubated with collagenase, pronase, and DNAse I at 37uC for 2 hr. The epithelial organoids were separated from the stromal cells and debris by iso-osmotic Percoll gradient centrifugation (500 x g, 30 min). After centrifugation, the stromal cells remained near the top of the gradient while the epithelial cell organoids banded at a higher density near the middle (r = 1.0521.07 g/ml) of the gradient. Epithelial organoids were collected and washed with M199 medium. The epithelial organoids were plated onto a culture dish that had been coated with 5 mg/ml mouse laminin (Sigma-Aldrich) in keratocyte-SFM medium (GIBCO) supplemented with 2% fetal bovine serum (FBS), epidermal growth factor (EGF), and bovine pituitary extract. Upon reaching confluence, cells were re-plated to a new collagen-coated plate at a split ratio of 1:2. To immortalize PECs, epithelial cells were infected with lentivirus expressing LT antigen. Two days after infection, epithelial cells were split 1:4 and selected with G418 (0.5 mg/ ml) for 2 weeks. The G418-resistant cells were expanded. BrdU Labeling of Cultured Cells For the BrdU incorporation assay, cells (50–70% confluent) were cultured on coverslips in the presence of BrdU (10 mM) for 8 hrs. The BrdU-labeled cells were detected employing a monoclonal anti-BrdU antibody (BD Biosciences, Franklin Lakes, NJ). Real-time PCR Male C57BL6/J mice (n = 20) at the age of 7, 14, 30, or 60 days were killed, and the whole prostate glands were dissected. Total RNA was isolated using the TRIzol Reagent and reverse transcribed using the Reaction Ready First Strand cDNA Synthesis Kit (SuperArray Bioscience Corp., Frederick, MD). The cDNA products were PCR- amplified (40 cycles of 30 s at 94uC; 20 s at 55uC; 30 s at 72uC) with the RT2 real–time SYBR green PCR master mix and the gene-specific primer sets for mouse genes encoding prostate-secreted proteins and b-actin genes (SuperArray Bioscience Corp.) in a SmartCycler II (Cepheid, Sunnyvale, CA). Raw data processing and quantification were performed with the SmartCycler software (version 2.0C) [40]. Immunohistochemistry Formalin-fixed, paraffin-embedded mouse prostate sections were deparaffinized by sequential washing with xylene, graded ethanol, and PBS. Antigen retrieval was done by heating the samples in a steam cooker in 16Target Retrieval Solution (Dako) for 30 min. After the samples were cooled and washed with PBS, endogenous peroxide was blocked with 3% hydrogen peroxidase inhibitor in PBS for 12 min. Nonspecific proteins were blocked by immersing the sections in 5% horse serum and 1% goat serum for 20 min. Slides were incubated with anti-p44 (1:400), -CK18, - CK5, -BrdU, or -Ki67 antibodies overnight at 4uC and then with a secondary peroxidase-labeled anti-rabbit antibody (1:500; Jackson ImmunoResearch) for 1 h at room temperature. Signal was detected by staining with 3,39-diaminobenzidine (DAB; Phoenix Biotechnologies) substrate for 6 min and then counter- staining with Gill’s hematoxylin No. 3 (Sigma) for 20 s. For cultured cells, cells were grown on chamber slides and fixed with cold methanol (220uC) for 10 min. Nonspecific proteins were blocked in 4% fish gelatin in PBS for 20 min. Overnight incubation at 4uC with primary antibodies was performed followed by a 1-hr incubation with goat anti-rat Alexa 595 (1:500; Invitrogen) at room temperature. After washes in PBS, the samples were counterstained with Sytox green (Molecular Probes) for 10 min at room temperature, mounted in Histogel (Linaris Histogel), and analyzed directly by fluorescence confocal micros- copy. PLOS ONE | www.plosone.org 13 November 2012 | Volume 7 | Issue 11 | e49173