p44 Functions in Prostate Epithelial Cells Figure 4. Cytoplasmic p44 is essential for growth of prostate epithelial cells. (A) Expression of p44, NES-p44, or NLS-p44 in the p44-null prostate epithelial cells. Western blot analysis of whole cell lysates (10 mg) made from p44-null prostate epithelial cells infected with control lentivirus (lane 1) or lentivirus expressing p44 (lane 2), NES-p44 (lane 3), or NLS-p44 (lane 4), as indicated. The p44 wild-type (p44loxP/loxp) epithelial cells infected with control lentivirus were used as the control (lane 5). (B) The subcellular localization of p44, NES-p44, and NLS-p44 in prostate epithelial cells. Immunostaining of p44 in p44 wild-type (p44loxP/loxp) epithelial cells (top panels), in p44-null prostate epithelial cells infected with control lentivirus (2nd panel) or lentivirus expressing p44 (3rd panel), NES-p44 (4th panel), or NLS-p44 (bottom panel), as indicated. P44 and the nucleus were stained in red and green, respectively. The left pictures are a merger of p44 and nuclear staining. (C) Cytoplasmic p44 fully restored growth arrest of prostate epithelial cells induced by loss of p44. Growth curves of p44-null prostate epithelial cells infected with control lentivirus or lentivirus expressing p44, NES-p44, or NLS-p44. One-way ANOVA analysis: F = 43.00, p,0.0001, R square = 0.9485. The p value of unpaired Student’s t-Test of data between p44loxP/loxP and NES-p44, p44, or p442/2 epithelial cells are 0.3352, 0.2873, or 0.0003. doi:10.1371/journal.pone.0049173.g004 PLOS ONE | www.plosone.org 5 November 2012 | Volume 7 | Issue 11 | e49173