Extended Data Fig. 4: CLU⁺ cells are distinct from CBCs.

a, SSC2 t-SNE plots are overlaid with expression of the indicated genes (n = 313 single-cell transcriptomes). Clu^high cells are circled in each map (black circles) and cells co-expressing either Lgr5 (middle) or Ascl2 (right) are shown in green. No CLU⁺ cells co-expressing Lgr5 were observed, and CLU⁺ cells co-expressing Ascl2 were rare (green circles with green arrows, right). Gene-expression values of the indicated genes are shown as log₁₀(CPM). b, Representative images and close-up views (yellow box and bottom panels) are shown of smFISH analyses of endogenous Clu (green) and Lgr5 (red) mRNA expression in the small intestine, counterstained with DAPI (blue) at the indicated times after irradiation. Note that CLU⁺ cells (green arrowheads) are distinct from LGR5⁺ cells (red arrowheads) with occasional examples of CLU⁺ cells at 3 dpi that contain Lgr5 transcripts highlighted with yellow arrowheads (n = 2 mice each). Analyses of non-irradiated intestines is shown in Fig. 2a. c, Endogenous Clu mRNA identified by smFISH (yellow) followed by BAC-Clu-GFP transgene visualization by immunostaining (green) is shown in irradiated small intestine. White arrowheads indicate cells co-expressing Clu and GFP (n = 1 mouse). d, Immunohistochemistry analysis of Clu-GFP⁺ cells in small intestine of untreated (0 Gy) or irradiated (2–4 dpi) mice (n = 2 mice each). e, Either Lgr5-GFP⁺ or Clu-GFP⁺ cells within the EPCAM⁺ epithelial cell fraction of small intestine were quantified by flow cytometry (see Extended Data Fig. 5b) and are plotted as percentage at the indicated dpi (n ≥ 2 mice each). f, Immunostaining of Clu-GFP (green, yellow arrowheads) and OLFM4 (red, white arrowheads) expression in the crypt regions of small intestine. (n = 2 mice). Scale bars, 35 μm (b); 25 μm (c); 40 μm (d); 25 μm (f).

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