Fig. 3.

Steroid receptor expression in d-7 prostaspheres and growth response of progenitor cells to E₂. A, Steroid receptor mRNA levels in d-7 prostaspheres, LNCaP, and PC3 cell lines as determined by quantitative real-time PCR. Data are normalized to LNCaP levels (set as 1) after normalization of each sample to GAPDH. Although the prostate progenitor cells did not express AR, there was robust expression of ERα, ERβ1, GPR30, and PR. ERβ expression was measured using primers specific for ERβ1 as well as primers that amplify all ERβ isoforms (ERβall); n = 3 separate experiments. B, Immunofluorescent labeling of d-7 prostasphere cells for ERα, ERβ, or GPR30. ERα and ERβ primarily localized to the nucleus, whereas GPR30 localized to the cell membrane and, to a lesser degree, cytoplasm. The inset shows fluorescence with IgG substituted for primary antibody. Scale bar, 50 μm. C, Prostaspheres were grown for 7 d in a 3D matrigel culture in the absence or presence of 1 nm E₂, and prostasphere numbers and sizes (40–80 μm, >80 μm diameter) were measured. Both the number of prostaspheres that formed as well as their size at d-7 were markedly increased in response to estrogen exposure. *, P < 0.05 vs. control; n = 6 separate experiments.