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. 2019 Apr 10;21(9):2103–2115. doi: 10.1038/s41436-019-0476-3

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© The Author(s) 2019

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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Fig. 4 — ALPK1 localization to the centrosomes and base of the cilia in cells and the impact of ALPK1 (c.710C>T, [p.Thr237Met]) variant on primary cilia assembly. (a–c) Antibody staining of fixed ARPE19 cells with anti-ALPK1 (green, Proteintech, cat #19107-1-AP) and anti-α-tubulin (red) antibodies in a mitotic cell shows the localization of ALPK1 in spindle poles. (d–f) Further immunostaining of ARPE19 cells with anti-γ-tubulin (red) confirmed the centrosomal localization of ALPK1 during mitosis. (g–i) Localization of ALPK1 in ARPE19 cells in interphase shows a diffuse cytosolic pattern and enrichment of ALPK1 in centrosomes of the cells, which colocalize with γ-tubulin. (j–l) ARPE19 cells were serum starved for 24 hours and immunostained with anti-ALPK1 (green, Proteintech, cat #19107-1-AP) and anti-acetylated-α-tubulin (red) showing the ALPK1 localization at the base of primary cilium. Cell nuclei are stained with DAPI (blue). (m) Fibroblast cells were treated with serum-free medium for 24 hours and labeled with primary cilia markers anti-acetylated-α-tubulin (red), anti-IFT88 (green), and DAPI (blue). Representative images of unaffected cells and affected cells carrying ALPK1 (c.710C>T, [p.Thr237Met]) variant show decreased number of ciliated cells in fibroblasts from affected patients compared with control. (n) Quantification of the ciliated cells in one unaffected control and two affected patients. The analysis showed significant decrease in assembly of primary cilia in the affected patients carrying ALPK1 (c.710C>T, [p.Thr237Met]) variant. The graphs show the means ± s.e.m. from three independent experiments where 100 cells were scored for each sample in each experiment. *p<0.05. Scale bars represent 5 μm in (a, d, g, j) and 10 μm in (m, n).