Figure 3. Phenotypic characterization of NHPrE1 and BHPrE1 cells. A: Immunoﬂuorescence staining of NHPrE1 and BHPrE1 cells shows 100% immunoreactivity to a WS-CK, conﬁrming the epithelial nature of the cells. NHPrE1 cells show expression of CK14 in most cells, and b- catenin is seen at the membrane interface of adjacent cells while 15-LOX-2 is detected in the cytoplasm. Consistent with the CK14 data, nuclear p63 is present in most cells, as are CD133 and CD44. OCT-4 staining is variable but detectable in most cells. BHPrE1 cells are also epithelial with all cells exhibiting immunoreactivity to WS-CK and many to CK14 but with lower expression of CD133, CD44, and OCT-4. 15-LOX-2 is detected in a minority of cells. Scale bar ¼ 50 lm. B: Western blotting shows that CD133, CD44, and PTEN proteins are expressed at elevated levels; however, SHH and RB proteins are expressed at decreased levels in NHPrE1 cells compared to BHPrE1 cells. In contrast, NKX3.1 and the cell cycle–regulating protein p53 are similarly expressed in BHPrE1 and NHPrE1. A moderate level of TERT is shown in both NHPrE1 and BHPrE1 cells compared to NHPrE0 and BHPrE0 cells, respectively. BHPrE1 shows a low level of p16 protein expression compared to BHPrE0. In contrast, NHPrE1 shows enhanced p16 protein compared to NHPrE0. Abbreviations: 15-LOX-2, 15-lipoxygenase-2; BHPrE0, primary cultured benign human prostate epithelial cells isolated from a prostate surgical sample; BHPrE1, a spontaneously immortalized benign human prostate epithelial cell line from BHPrE0; NHPrE0 (NHP8), primary cultured ‘‘normal’’ human prostate epithelial cells isolated from a donated, 41-year- old, healthy, male prostate; NHPrE1: a spontaneously immortalized ‘‘normal’’ human prostate epithelial cell line, that was established from parent NHPrE0 cells; WS-CK, wide-spectrum anti-keratin antibody.