Different Smo agonists and antagonists affect Smo activities, differently. (A) Immunofluorescence images of RPE1 cells plated at LCD and treated with different concentrations of Hh agonists and antagonists as indicated. (B) Quantification of ciliated cells at different conditions as indicated. Statistical significances compare all conditions to control cells with Shh. (C) Western blot probed for Smo indicating Smo KO in CRISPR cell line compared with RPE1 WT. (D) Graph indicating cAMP Glo sensor expression levels as an indicator for cAMP activity for each condition as indicated. (E) Graph indicating Gli1 expression levels for each condition as indicated in comparison to serum-starved ciliated cells. Statistical significance compares all conditions to serum-starved cells at HCD without Shh. *, P < 0.01; **, P < 0.001; ***, P < 0.0001, comparing cells with Shh to those without Shh for the same condition unless otherwise indicated. Number of cells (n) used for each experiment (three repeats) is listed in Table S4. Yellow and red numbers and arrows indicate the presence or absence of a cilium, respectively.