Smo agonists differentially activate LKB1 or Gα_i, and both are required for ciliogenesis. (A) Immunofluorescence images of RPE1 WT, control siRNA, Smo KO, LKB1-depleted, and Gα_{i (1+2+3)}–depleted cells at LCD and treated with Shh or different Smo agonists as indicated. (B) Quantification of ciliated cells at different conditions as indicated. Gα_i siRNA represents KD of different isoforms, Gα_{i (1+2+3)}, and OFD1 siRNA represents KD of a combination of siRNAs, OFD1_(1+2+3). (C) Western blots probed with LC3 or p62 to assess the activation of autophagy at different conditions as indicated. Cells were treated with bafilomycin A1 (BAF A1; 100 nM) for 2 h followed by an additional 6 h supplemented with Shh CM. Black lines separate nonconsecutive parts of the same sets of Western blots. (D) Quantifications of LC3II/LC3I ratio and p62 levels to measure the autophagy flux for Western blots shown in C. (E) Quantification of ciliated cells at different conditions as indicated. *, P < 0.01; **, P < 0.001; ***, P < 0.0001 comparing all conditions to control cells with Shh. 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.