Hh pathway agonists and antagonists stimulate Smo translocation to the primary cilium.

<p>(A) Wild-type mouse embryonic fibroblasts (MEFs) stained with antibodies against endogenous Smo (green), acetylated (AC) tubulin (red), and DAPI (blue). MEFs treated with ShhN-conditioned media (ShhN-CM), purmorphamine, cyclopamine, and jervine display Smo staining along the entire length of the cilium. Note that fixation with paraformaldehyde produces artifactual nuclear staining with the Smo antibody; this nuclear staining is not visible after fixation in methanol (See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005182#pone-0005182-g003" target="_blank">Fig. 3C</a>). (B) Quantification of the percentage of Smo-positive (Smo⁺) cilia in MEFs after treatment with ShhN CM and cyclopamine for the indicated times. Cyclopamine induces Smo translocation to a significant number of cilia after 6 hours of treatment, although this is less than that induced by ShhN CM. All quantification of cilia staining in this and subsequent figures represents at least two separate experiments with a minimum of 100 cilia scored per time point and condition. Error bars indicate +/− standard deviation (SD). (C) Quantification of Smo⁺ cilia in MEFs after treatment with indicated compounds for 24 hours. 20-α-hydroxysterol (20α-OHC), purmorphamine, cyclopamine, and jervine induce Smo translocation to the cilium. Error bars indicate +/− SD.