Extended Data Fig. 5: Nox1+ colon cells express markers of undifferentiated stem cells.

a) qPCR analysis on Nox1- and Npy1r-GFP FAC-sorted cells showed an enrichment in stem cell markers Lgr5 (Nox1 caecum n = 3, 12.42 ± 1.219; Nox1 colon n = 4, 14.0825 ± 1.656; Npy1r colon n = 6, 9.6516 ± 0.5590), Smoc2 (Nox1 caecum n = 3, 5.8765 ± 0.2463; Nox1 colon n = 4, 7.5078 ± 0.3144; Npy1r colon n = 4, 6.368 ± 0.7178), Ascl2 (Nox1 caecum n = 3, 7.613 ± 1.399; Nox1 colon n = 4, 17.916 ± 0.7981; Npy1r colon n = 4, 11.0515 ± 2.212) (upper panel). Lineage markers Dra (Nox1 caecum n = 4, 0.079 ± 0.03201; Nox1 colon n = 5, 0.18 ± 0.04231; Npy1r colon n = 7, 0.19 ± 0.03690), Muc2 (Nox1 caecum n = 3, 0.2367 ± 0.09939; Nox1 colon n = 3, 0.18 ± 0.06245; Npy1r colon n = 7, 0.09 ± 0.01952), ChgA (Nox1 caecum n = 3, 0.1667 ± 0.1217; Nox1 colon n = 3, 0.12 ± 0.04041; Npy1r colon n = 7, 0.7814 ± 0.1142), and Reg4 (Nox1 caecum n = 3, 0.15 ± 0.04807; Nox1 colon n = 3, 0.3967 ± 0.1530; Npy1r colon n = 7, 0.57 ± 0.1214) were upregulated in the GFP⁻ population, indicating that Nox1⁺ and Npy1r⁺ cells enrich for stem cells. Statistical significance was determined by two-tailed t-test; b-c) Co-IF for GFP and lineage markers using Nox1-2A-EGFP (n = 5) (b) and Npy1r-EGFP-IRES-CreERT2 (n = 5) (c) mice colon (caecum, distal colon and rectum) showed that GFP⁺ cells do not overlap with the lineage markers. Co-IF with Ki67 indicated that Nox1⁺ and Npy1r⁺ cells are proliferative. Scale bar: 25μm.

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