Differentiation status and AR expression profile of PCSCs under the influence of Hedgehog overexpression. The boxed areas in the pictures are further magnified and shown in the corresponding lower or right pictures. (A) Increased CK14+ cells along with tumorigenic progression in the pCX-shh-IG-injected prostate ((b), (c) and (d)), as compared to the normal prostate ((a)). (B) Western blot analysis indicated up-regulation of CK14, CD44 and CK8 in the pCX-shh-IG-injected prostates as compared to the normal pCX-IG-injected prostates. (C) Characterization of PCSCs by double-immunofluorescence staining showing differentiation toward CK14+ progeny ((a), (b) and (c)), CK14+ cells toward CD44+ progeny ((d), (e) and (f)), and CK14+ cells toward CK8+ progeny ((g), (h) and (i)). CK14+ differentiation involved Hedgehog signalling activation, as indicated by co-localized Patch1 expression ((j), (k) and (l)). The basal-intermediate (CK14+/CD44+) and intermediate-luminal (CK14+/CK8+) populations were increased in PIN and CaP of pCX-shh-IG-injected prostates as compared to those of the pCX-IG-injected vehicle controls (D and E). (F) Some PCSCs were AR- as indicated by arrows in (a), (b), (c), (d), and (e), even though they were P63+, CK14+, CK8+, CD44+, or Patch1+. The relative proportions of AR+ and AR- cells among P63+, CK14+, CK8+, CD44+, and Patch1+ cell populations were shown respectively in (f). All scale bars represent 10 μm in length. CaP: prostate cancer; PIN: prostatic intraepithelial neoplasia; HGPIN: high grade prostatic intraepithelial neoplasia.