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. 2017 Jul 4;8:15857. doi: 10.1038/ncomms15857

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Copyright © 2017, The Author(s)

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/

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(a) E2f4-Pcm1 IF confocal image, stage1-2 transition during E2f4 nucleocytoplasmic shift: Pcm1-E2f4 signals adjacent but still not overlapping (inset, enlarged from circled area). (b) Left panel: confocal image stage 2 cells showing apical Pcm1-labelled granules nearby primary cilia (acetylated α-tub); middle panel: 3D-SIM: Pcm1-E2f4 colocalization (circled area representative of apical granule on left): E2f4 at the core of Pcm1 granules); right panel: confocal image depicts Pcm1 underneath and nearby Gt335-labelled primary cilium. See also Supplementary Movie 1. (c) IF confocal (upper panel) and 3D-SIM (lower panel) of stage 1–2 cells: E2f4 nucleocytoplasmic translocation and initial association with Deup1 (inset, enlarged from circled area). Assembly of Deup1, Cep152 and cytoplasmic E2f4 into deuterosomes. (d) Proximity ligation assay (PLA): Deup1+E2f4 proximity signal overlapping with Pcm1 (left and split panels: circled areas in ALI day3 from E2f4^f/f; R26^CreERT2/+ no Tm administration); specificity of signals confirmed in E2f4-deficient cells (right panel; Tm-treated E2f4^f/f; R26^CreERT2/+ cultures; see also Supplementary Fig. 4a). (e) Co-immunoprecipitation (coIP) assays: binding of endogenous (top) or exogenous (bottom) E2f4 to Deup1 in ALI day 3 (stage 2) or Deup1-Flag in 293FT homogenates, respectively (see methods). (f) Deup1-E2f4 IF, 3D-SIM reconstruction of stage 2 cells (top left) and cells transitioning to stage 3 (top right). Small Deup1 dots assemble into rings inside E2f4 granules but, later, more mature deuterosomes with larger rings locate at the periphery or outside the E2f4 granules. Arrows i,ii,iii: representative Deup1 rings from circled areas enlarged in lower panels shown by split channel (E2f4) or double Deup1-E2f4 labelling (less to more mature deuterosomes from left to right). Bars: a–d,f=5, 1, 0.5, 5, 0.5 μm, respectively.