Noncanonical Hh promotes ciliogenesis through Smo activity. (A) Immunofluorescence images of RPE1 cells at LCD supplemented with serum and treated with Shh CM (24 h) containing different concentrations of Shh ligand as indicated. (B) Quantification of Shh ligand concentrations and percentage of ciliated cells for each Shh CM as indicated. (C) Immunofluorescence images of RPE1 cells plated at LCD supplemented with serum and treated with Shh CM (i), immunodepleted Shh CM (Imm-Dep Shh CM; ii), CM cultivated from nontransfected HeLa cells (HeLa CM; iii), CM cultivated from nontransfected HeLa cells and immunodepleted for Shh (Imm-Dep HeLa CM; iv), and Shh CM with mock immunodepletion (Mock-Dep), as indicated (v). (D) Quantification of ciliated cells for each condition as indicated. (E) WT and Smo KO RPE1 cells transfected with full-length Smo-GFP and SmoΔC-GFP. Cells were grown in serum, with or without Shh. (F) Quantification of ciliated cells for each condition as indicated. *, P < 0.01; **, P < 0.001; ***, P < 0.0001. Number of cells (n) used for each experiment (three to four repeats) is listed in Table S4. Yellow and red arrows indicate the presence or absence of a cilium, respectively.