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. 2021 Mar 26;4:417. doi: 10.1038/s42003-021-01941-5

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© The Author(s) 2021

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. 1 — a Scheme for the generation of doxycycline (Dox) inducible CENP-A overexpression cell lines. TetOn-CENPA-FLAG-HA construct was randomly integrated into several indicated target cell lines by lentiviral transfection without selection. After clonal isolation, we tested cells for clear homogenous CENP-A and HA increase by IF in approximately all cells 24 h after the addition of 80 ng/ml of Dox. Cells were also tested to ensure no detectable background HA signal by western or IF, when no Dox was added. b Scheme representing relative CENP-A protein levels over time for TetOn-CENPA-FLAG-HA cell lines. Dox = 10 ng/ml. c Western blot of total cell extracts (TCEs) pertaining to scheme in (b). Primary antibodies indicated on the right. Exogenous CENP-A (tagged) can be distinguished from endogenous CENP-A (endogen.) by its increased molecular weight. # = high sensitivity ECL. y-tubulin used as loading control. See also Supplementary Fig. 1B for western blot analysis corresponding to all cell lines. d Immunofluorescence of paraformaldehyde-fixed cells using anti-CENP-A antibody (green) and DAPI staining (gray). Conditions in parallel with (c). Showing max projection images from a Z-series with zoom on a mitotic cell (DAPI/CENP-A merge) to highlight chromosome arms. Scale bars = 10 µm, Zoom = 2.5 µm.