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. 2023 Dec 20;14:8484. doi: 10.1038/s41467-023-44138-6

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© The Author(s) 2023

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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 4 — a Schematic representation of BrdU injections (green) in NMRs and time of colonic tissue collection (red). b Images of NMR colonic crypt stained with anti-BrdU antibody (brown) and haematoxylin (blue). CBC cells are marked by dotted circles. c Bar graphs showing the mean percentage (±SEM) of labelled CBC cells (BrdU⁺CBC) over time, derived from counting n = 30 crypts per animal (3 NMRs sacrificed at 0.33, 1, 2 days; 2 at 0.67, 3, 4, 5 days). d Confocal image showing Lgr5 mRNA (red dots), BrdU (green) and DAPI (blue) in NMR colonic crypt cells after 3 days of BrdU injection. Asterisks (*) mark Lgr5⁺ cells at the crypt base (Lgr5^+CBC). White arrows indicate Lgr5^+CBCBrdU⁺ cells. e Scatter plot showing a linear increase in the BrdU labelling index (Number of Lgr5^+CBCBrdU⁺/Total number of Lgr5^+CBC) over time in NMRs. Each dot represents a mean of 40 crypts (±SEM) counted per animal (n = 3 animals at 0.33, 1 and 2 days; n = 2 animals at 0.67, 3, 4 and 5 days). f Top, Schema showing times when BrdU was injected (green) and colons harvested (red) in mice. Bottom, scatter plot showing a linear increase in the labelling index of murine Lgr5^+CBC cells with time. Each dot denotes the mean (±SEM) of 30 crypts counted per animal (n = 3 animals/time point). g Bar graphs showing the duration of specific cell cycle phases of Lgr5^+CBC cells in mice and NMRs. Exact P-values are indicated on the graphs. h Experimental design for measuring the total cell cycle time (T_T) of Lgr5⁺ cells above the crypt base by injecting BrdU (green) in mice and NMRs and time of tissue collection (red). i Bar graphs showing T_T of colonic Lgr5⁺ cells above the crypt base (left) and Lgr5^+CBC cells (right) in mice and NMRs. Exact P-values are indicated on the graphs. j Stacked bar graphs showing the time (days) spent by mouse, human and NMR colonic ASCs in each phase of the cell cycle. Student’s t-tests using two-tailed, unpaired and unequal variance were used. Scale bars are indicated on the images (25 µm).