Hh activates autophagy to promote ciliogenesis. (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 LC3 to assess the activation of autophagy and acetylated (Ace) Tubulin as a ciliary marker. Torin1 concentration is 250 nM. Cells were treated with Torin1 or Shh CM for 24 h. (B) Quantification of average of LC3 puncta/cells for each condition as indicated. Statistical significance compares all conditions to serum-starved cells at HCD. (C) Western blots were probed with LC3 or p62 to assess the activation of autophagy at different conditions as indicated. Cells were treated with Torin1 and bafilomycin A1 (BAF A1; 100 nM) for 4 and 2 h, respectively, followed by an additional 24 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 for each siRNA depletion 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, red, and green arrows point to cilium, no cilium, and LC3 puncta, respectively.