2.6.1 Imaris 3D reconstruction and parametric analysis

The measurements were carried out on URECs at passages 2–3. Z-stack images were acquired using an Olympus FV10i confocal laser scanning microscope and processed with Imaris 7.4.2 (Bitplane, UK) for 3D reconstruction. The volume and sphericity of the nucleus (DAPI), cilia (Acet-Tub and ARL13B), and pericentrin (PCNT) were automatically calculated based on Z-stacks composed of individual images. The cilia axis length was then calculated using three parameters (A, B, and C) multiplied by 2, following the manufacturer’s instructions (http://www.bitplane.com/download/manuals/ReferenceManual9_2_0.pdf). Axis A was the length of the shortest principal axis, axis B was the length of the second longest principal axis, and axis C was the length of the longest principal axis (Supplementary Figure S1). The analysis included a minimum of 100 cilia per individual, and the cilium frequency analysis included 100 cells per individual.