Ciliary assembly/disassembly assay

The cilium assembly and disassembly assays were carried out as previously described 20. Briefly, RPE‐1 cells (ATCC CRL‐4000, gift from Steve Doxsey) were serum‐starved for 36 h to induce ciliogenesis; serum was then added back to the medium to trigger ciliary resorption. For chemical treatment, CytoD (0.5 μM in DMSO), dynasore (80 μM in DMSO), or DMSO was included in the medium in the last 1 h of starvation as well as after serum re‐addition until the time of harvest. Jasplakinolide (20 nM in DMSO, Merck) was included in the last 15 min of starvation and thereafter. In some experiments, cells were transfected using Nucleofector (Lonza) or Neon transfection system (Thermo Fisher Scientific), and subjected to serum starvation ~16 h after transfection for ciliary assembly/disassembly. Cells transfected with more than one plasmid were routinely immunostained 24 h post‐transfection to confirm that the co‐transfection efficiency was > 90%. The number of cells expressing Ac‐Tub (see staining protocol below) was counted under epifluorescent microscopy in a double‐blind fashion. In the experiments involving transfection, only transfected cells expressing GFP (encoded by one of the plasmids) were counted. More than 100 cells were counted in each experiment, and at least three independent experiments were performed for each condition. Confocal microscope images were acquired with a 63× objective on either a Leica TCS SP2 spectral confocal system or Zeiss LSM 780 confocal microscope enabling the acquisition of images at the z‐interval of 0.2 μm.