Confirmation of Palbociclib activity and depletion ofautophagy proteins by siRNA. (A) Immunofluorescence images of RPE1 Geminin:GFP cells treated with Palbociclib (200 nM) for 24 h to arrest the cell cycle. Geminin, the DNA replication inhibitor, is absent in G1 phase and is highly expressed at the beginning of S phase through G2. Using Palbociclib, cells were Geminin negative showing G0/G1 arrest. (B) Immunofluorescence images of RPE1 cells treated with Palbociclib (200 nM) for 24 h to arrest the cell cycle at G0/G1 phase. Arl13B was used as a ciliary marker. (C) Stitched images of RPE1 Geminin:GFP cells treated with or without Shh at LCD and stained for GFP, indicating cells expressing nuclear GFP signals. White arrows indicate the presence of Geminin in nucleus. (D) Quantification of cells expressing nuclear Geminin at different conditions as indicated. (E) RPE1 cells grown in serum, with or without Shh, after depletion of ATG5 as an example for autophagy genes siRNA screen. (F) Western blots probed for different autophagy proteins and LC3, indicating the efficiency of each KD and its effect on autophagy flux in RPE1 cells. Number of cells (n) used for each experiment (three repeats) is listed in Table S4. Yellow and red arrows indicate the presence or absence of a cilium, respectively.