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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. 3 — a Schema showing BrdU injection schedule in NMRs (green) and time of tissue collection (red). b NMR crypts co-stained with anti-BrdU antibody (brown) and haematoxylin (blue). Dotted circles indicate crypt-based columnar (CBC) cells. c Bar graphs showing the mean percentage (±SEM) of labelled CBC cells (BrdU⁺CBC) at different time points in NMRs, derived by counting n = 20 crypts per animal (3 animals sacrificed at 0.33, 1, 2 days and 2 at 0.67, 3, 4, 5 days). d Immunofluorescent image showing Lgr5 mRNA (red), BrdU (green), and DAPI (blue) in NMR crypt injected with BrdU for 3 days. Asterisks (*) mark Lgr5⁺ cells at the crypt base (Lgr5^+CBC) and white arrows indicate Lgr5^+CBCBrdU⁺ cells. e Scatter plot showing a linear relationship between the ratio of Lgr5^+CBCBrdU⁺ cells to the total number of Lgr5^+CBC cells (labelling index) with time in NMRs until BrdU labelling becomes saturated. Each data point represents the mean (±SEM) of 20 crypts counted per animal (n = 3 animals at 0.33, 1 and 2 days; n = 2 at 0.67, 3, 4 and 5 days). f Top, BrdU injection times in mice (green) and time points when small intestinal tissues were collected (red). Bottom, scatter plot showing a linear increase in BrdU labelled 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 (mean ± SEM) showing the length of specific cell cycle phases of mouse and NMR Lgr5^+CBC cells. Exact P-values have been indicated on the graphs. h Schema showing the experimental design for estimating the total cell cycle time, T_T, of Lgr5⁺ cells located above the crypt base by injecting BrdU (green) in mice and NMRs and time points when tissues were collected (red). i Bar graphs showing the mean (±SEM) T_T of Lgr5⁺ cells above the crypt base (left) and Lgr5^+CBC (right) in mice and NMRs. Exact P-values have been indicated on the graphs. Statistical significance was determined by performing Student’s t-tests using two-tailed, unpaired and unequal variance. Scale bars are indicated on the images (25 µm or 50 µm).