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. 2018 Feb 14;8:3019. doi: 10.1038/s41598-018-21329-6

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© The Author(s) 2018

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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Kinesin 1 requires CCDC28B to regulate cilia length. (A) The proportion of cilia-positive cells is significantly reduced upon CCDC28B KD but not KLC1 KD (hypothesis test for proportions). Co-transfection of the KLC1 stealth oligo rescues the phenotype of CCDC28B KD cells. (B) CCDC28B KD (118 analyzed cilia) and KLC1 KD (155 cilia measured) cells show significantly shortened and elongated cilia respectively. Cilia were of control length in cells co-transfected with both stealth oligos (115 cilia). Statistical test: one-way ANOVA; ***P < 0.001. The amount of transfected oligos per condition was maintained constant using a control stealth oligo. (C) CCDC28B CRISPR clone B1 was analyzed by immunofluorescence and confocal microscopy using anti-acetylated tubulin (red), anti γ-tubulin (green) and DAPI (blue) to stain cilia, basal bodies and nuclei respectively. Untreated hTERT-RPE cells were used as control and CCDC28B CRISPR clone B1 transfected with pCS2+ _CCDC28B wt was used to show rescue and specificity of the cilia phenotype. (D,E) Clone B1 cells were analyzed to quantify the number of total cilia-positive cells (acetylated α-tubulin signal irrespective of length) and cells bearing “long” cilia of at least 1 μm. Scale bars represent 10 μm. (E) Quantification shows that while the total number of cilia is similar between hTERT-RPE controls and Clone B1, the later presents significantly less cilia of at least 1 μm. Importantly, the proportion of cells bearing 1 μm cilia is rescued upon transfecting wt CCDC28B. 80 cells were analyzed for control and clone B1 cells, and 70 cells were scored for clone B1 overexpressing CCDC28B. Statistical test used: hypothesis test for proportions. Data are representative of three independent experiments. (F) Genetic interaction experiment using CCDC28B CRISPR clone B1. KLC1 KD and KIF5ABC KD result in a mild rescue (compared to that of KIF7) that is abolished or diminished by further CCDC28B KD for KLC1 and KIF5s respectively. KIF7 KD rescues the ciliary phenotype irrespective of the presence of CCDC28B. The experiment shown is representative of four independent experiments. The total number of cells scored for this experiment is 89 Ctrl.-Ctrl., 96 Ctrl.-CCDC28B, 91 Ctrl.-KLC1, 86 KLC1.-CCDC28B, 84 Ctrl.-KIF5ABC, 86 KIF5ABC-CCDC28B, 84 Ctrl.-KIF7, 104 KIF7.-CCDC28B. Statistical test: hypothesis test for proportions. *Indicate comparison to Clone B1 transfected with control oligos (S.CTRL-S.CTRL). ^♦Indicate comparison to Clone B1 transfected with control oligo and CCDC28B oligo (S.CTRL-S.CCDC28B). ^*/♦P < 0.05; ^**P < 0.01; ^{***/♦♦♦}P < 0.001. Scale bars correspond to 10 μm.