Fig. 2.

A, Immunofluorescent labeling of d-7 prostaspheres with Nanog, TROP2, CD49f, SSEA4, and ABCG2 confirms their stem/early progenitor cell characteristics. The majority of prostasphere cells were positive for membrane-associated TROP2, CD49f, ABCG2, SSEA4 [with blue 4′,6-diamidino-2-phenylindole (DAPI) counterstain], and nuclear Nanog. Confocal images are shown for TROP2, CD49f, and ABCG2, whereas fluorescent imaging of whole-mounted prostaspheres was employed for Nanog and SSEA4. In the latter, excess signal in the prostasphere center is a result of the dense cell compaction in the spheroid. Scale bar, 50 μm. B, Photomicrographs showing Hoechst exclusion by d-7 prostasphere cells and 2D PrEC in the absence or presence of 50 μm verapamil. Note that the exclusion of 0.5 μg/ml Hoechst dye by prostasphere cells (upper left) was blocked by verapamil which caused the retention of Hoechst dye (upper right), indicating stem/progenitor cell nature of the cells. In contrast, no significant Hoechst dye exclusion was found in the 2D culture PrEC cells (lower left), and there was no effect of verapamil on their dye retention (lower right). Scale bar, 50 μm.