Hh activation induces ciliogenesis at LCD and in the presence of serum. (A) RPE1 cells plated at different cell densities and different conditions as indicated. The number of cells plated/cm² is indicated. Cells were stained for acetylated (Ace) Tubulin as a ciliary marker. (B) Quantification of ciliated cells at different cell densities. (C) Hh-mediated cilia indicating different ciliary markers localized at cilia. (D) Immunofluorescence images of human fibroblast cells at LCD and treated with Shh (24 h). (E) Quantification of human fibroblast ciliated cells at LCD upon Shh treatment. (F) Immunofluorescence images of neurons from mouse cortex indicating a big field of view (stitches), as well as a higher magnification for ciliogenesis in cells with or without Hh activation. Hh was activated using either Shh CM or dual treatment of SAG plus GSA-10 for 24 h. (G) Quantification of ciliated cells at different conditions as indicated. **, P < 0.001; ***, P < 0.0001 comparing cells with Shh to those without Shh for the same condition. Number of cells (n) used for each experiment is listed in Table S4. Yellow and red numbers and arrows indicate the presence or absence of cilium, respectively.