Hh-dependent recruitment of SuFu and Gli proteins to cilia requires active Smo. (A) NIH-3T3 cells were treated with the Smo agonist SAG or with the antagonist Cyc. SuFu and Gli are recruited to cilia by SAG but not by Cyc, although both SAG and Cyc recruit Smo to cilia. The tips of cilia point to the left. Bar, 2 µm. (B) Cilia counts for the experiment in A. (C) Q-PCR assay of Hh pathway target genes for the experiment in A. (D) Maintaining increased levels of SuFu and Gli at cilia is continuously dependent on active Smo. Cyc was added in the presence of Shh to NIH-3T3 cells prestimulated with Shh for 3 h. Ciliary localization was determined before and after 3 h of Shh stimulation and 1 and 3 h after Cyc addition. (E) NIH-3T3 cells were stimulated with Shh for 3 h followed by incubation with the Smo antagonist SANT-1 for 3 h. Ciliary localization of SuFu, Gli, and Smo was measured at the indicated times. P < 0.002 for the recruitment of Smo, SuFu, and Gli by Shh stimulation. P-values for exit from the cilium were calculated relative to ciliary localization after 3 h of Hh stimulation. Asterisks indicate the p-values for ciliary exit (*, P < 0.05; **, P < 0.01; ***, P < 0.001). Error bars indicate mean ± SD.