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. 2020 Oct 21;10(10):200227. doi: 10.1098/rsob.200227

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© 2020 The Authors.

Published by the Royal Society under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/4.0/, which permits unrestricted use, provided the original author and source are credited.

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Figure 3. — CENP-A assembles in the canonical G1 phase of the pluripotent stem cell cycle. (a) SNAP-tag based quench-chase-pulse labelling: CENP-A-SNAP expressing hESC or HeLa cells were labelled with the non-fluorescent substrate (BTP; quench) followed by a chase period (5 h 30 min) during which new unlabelled protein is synthesized. Nascent protein is subsequently fluorescently labelled with TMR-Star (Pulse). Localization and fate of nascent fluorescently labelled CENP-A-SNAP is determined by high-resolution microscopy. (b) Representative fluorescence images of differentiated (HeLa-CENP-A-SNAP) cells or hESCs (hESC CENP-A-SNAP) as processed according to (a). Tubulin staining was used to identify midbodies, indicative of G1 phase cells. CENP-A-SNAP assembly occurs in a subset of cells and all midbody positive cells are positive for nascent CENP-A assembly. Arrows indicate cells in G1. Scale bar = 5 µm.